ORIGINAL
RESEARCH
Activation of Basal Gluconeogenesis by Coactivator
p300 Maintains Hepatic Glycogen Storage
Ling He, Jia Cao, Shumei Meng, Anlin Ma, Sally Radovick, and
Fredric E. Wondisford
Divisions of Metabolism (L.H., J.C., S.M., A.M., F.E.W.) and Endocrinology (S.R.), Departments of
Pediatrics, Physiology and Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland
21287
Because hepatic glycogenolysis maintains euglycemia during early fasting, proper hepatic glycogen synthesis in the fed/postprandial states is critical. It has been known for decades that gluconeogenesis is essential for hepatic glycogen synthesis; however, the molecular mechanism remains
unknown. In this report, we show that depletion of hepatic p300 reduces glycogen synthesis,
decreases hepatic glycogen storage, and leads to relative hypoglycemia. We previously reported
that insulin suppressed gluconeogenesis by phosphorylating cAMP response element binding
protein-binding protein (CBP) at S436 and disassembling the cAMP response element-binding
protein-CBP complex. However, p300, which is closely related to CBP, lacks the corresponding S436
phosphorylation site found on CBP. In a phosphorylation-competent p300G422S knock-in mouse
model, we found that mutant mice exhibited reduced hepatic glycogen content and produced
significantly less glycogen in a tracer incorporation assay in the postprandial state. Our study
demonstrates the important and unique role of p300 in glycogen synthesis through maintaining
basal gluconeogenesis. (Molecular Endocrinology 27: 1322–1332, 2013)
he liver plays a critical role in maintaining blood glucose levels within the normal range throughout the
fed-fast cycle. During early fasting, hepatic glycogenolysis
maintains euglycemia. In contrast, gluconeogenesis plays
a dominant role in maintaining blood glucose levels during prolonged fasting. During fed and postprandial states,
elevated blood glucose levels promptly increase insulin
and decrease glucagon secretion. These hormonal
changes act in concert to decrease glucose production in
the liver by suppressing glycogenolysis and gluconeogenesis and increase glucose utilization in peripheral tissues
by activating glycolysis. Glycogen metabolism is under
hormonal regulation by both glucagon and insulin (1–3).
Glucagon stimulates the breakdown of glycogen through
activation of glycogen phosphorylase by phosphorylating
this enzyme at Ser14 (4). In contrast, insulin increases
glycogen synthase (GS) activity through the activation of
protein kinase B (Akt), which subsequently leads to phos-
T
phorylation and deactivation of glycogen synthase kinase
3 (5–7). The phosphorylation of GS by glycogen synthase
kinase 3 at a cluster of COOH-terminal serine residues
inhibits GS enzymatic activity. Insulin also regulates glycogen metabolism through activation of phosphoprotein
phosphatase 1, which, in turn, mediates the dephosphorylation of GS and glycogen phosphorylase; these insulin
effects lead to the further activation of GS and inhibition
of glycogen phosphorylase (6). Conversely, glucose
6-phosphate plays a key role in regulating GS activity and
is able to negate the inactivation of GS due to phosphorylation and fully restore enzymatic activity (6, 8). The
increase in hepatic glucose 6-phosphate concentration
leading to an elevation in glycogen synthesis can be observed in prolonged (72 h) fasted mice (9).
Insulin also suppresses hepatic gluconeogenesis by
phosphorylating cAMP response element (CRE)-binding
protein (CREB)-binding protein (CBP), CRTC2, and
ISSN Print 0888-8809 ISSN Online 1944-9917
Printed in U.S.A.
Copyright © 2013 by The Endocrine Society
Received December 26, 2012. Accepted June 7, 2013.
First Published Online June 14, 2013
Abbreviations: Akt, protein kinase B; CBP, CREB-binding protein; CRE, cAMP response
element; CREB, CRE-binding protein; GS, glycogen synthase; shRNA, short hairpin RNA;
WT, wild type.
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Mol Endocrinol, August 2013, 27(8):1322–1332
doi: 10.1210/me.2012-1413
doi: 10.1210/me.2012-1413
FoxO1, leading to the disassembly of the CREB-CBP
complex (10 –12). In addition, CRTC2 and FoxO1 are
exported from the nucleus after their phosphorylation
and subjected to cytoplasmic degradation. These effects
of insulin action lead to suppression (⬃60%) of glucose
release and storage of glucose as glycogen (1, 13).
Reports from decades ago have suggested that the gluconeogenic pathway accounted for 50%–70% of newly
synthesized glycogen (14). In the perfused rat liver or in
primary rat hepatocytes, physiologic concentration of
glucose had minimal effect on the glycogen synthesis
when glucose was the sole substrate; however, efficient
glycogen synthesis occurred when gluconeogenic precursors were added (15, 16). Studies from rat, mouse, and
dog using radiotracer-labeling techniques have firmly established that the gluconeogenic pathway contributes
substantially to hepatic glycogen formation during postprandial state (17–22). Human studies reached the same
conclusion (23–26). Data from these studies indicate that
a significant amount of gluconeogenesis still occurs even
in the presence of elevated blood glucose levels and that
physiologic hyperinsulinemia does not completely inhibit
net gluconeogenic flux (17–27). Therefore, hepatic gluconeogenesis during the postprandial state has important
implications for converting gluconeogenic precursors,
such as lactate, fructose, and amino acids delivered from
the gastrointestinal tract and other tissues, into glucose
that can be stored as glycogen or released into blood as
glucose. In fact, 15% of the glucose uptake by muscle is
released as lactate, and lactate is then used in the liver to
synthesize glycogen through Cori cycle (28).
However, the underlying mechanism of glycogen synthesis through the gluconeogenic pathway in the postprandial state has not been elucidated. Recently, we reported that coactivator p300 maintains basal
gluconeogenesis in the fed and postprandial states (29).
Therefore, we investigated the possible role of p300 in
mediating glycogen synthesis. In the current study, we
found that p300 is critical and unique in its ability to
maintain hepatic glycogen synthesis in both the fed and
postprandial states.
Materials and Methods
Adenoviruses
The BLOCK-iT adenoviral RNA interference expression system (Invitrogen, Carlsbad, California) was used to construct
adenoviral short hairpin RNA (shRNA) for CBP, p300, CRTC2,
and scrambled shRNA as previously described (12, 29).
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Glucose production, glycogen synthesis, and
glucose uptake assays
Mouse primary hepatocytes from fed mice were cultured in
William’s medium E supplemented with insulin-transferrin-selenium (BD Biosciences, Palo Alto, California) and dexamethasone. Glucose production assays were performed as previously
described (12, 29). Primary hepatocytes were infected 16 –24
hours after planting with adenoviral shRNAs. After incubation
for 72 hours, cells were washed twice with PBS. For the glycogen
synthesis assay, the medium was replaced with 2 mL of glycogen
synthesis buffer consisting of 20 mM glucose and/or 20 mM
sodium lactate and 2 mM sodium pyruvate, and supplemented
with 20 nM insulin. After a 5-hour incubation, cells were
washed 5 times with PBS and collected, and then subjected to 3
freeze-thaw cycles. The glucose and glycogen readings were normalized to the total protein concentration. To measure the rate
of glucose uptake, primary hepatocytes from both wild-type
(WT) and p300G422S mice were subjected to serum starvation
in Williams E medium (11.1 mM glucose) without serum for 4
hours, after which cells were incubated in fresh William E medium supplemented with 1 ␮Ci/mL [2-3H]deoxyglucose
(PerkinElmer Life Sciences, Wellesley, Massachucetts) for 10
minutes. Reactions were terminated by 3 washes with ice-cold
PBS. Cells were collected and lysed in Lysis Buffer (Cell Signaling Technology, Danvers, Massachusetts). After centrifugation
at 3000 rpm for 10 minutes, a portion of the lysate was used for
the measurement of protein concentration; the remainder was
used for scintillation counting, with results expressed as counts
per minute in the cell lysate/mg protein.
Animal experiments
All animal protocols were approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University.
Adenoviral shRNA knockdown experiments were conducted
64 –72 hours after mice were injected with the adenovirus (12,
29). Because mice have a very long period of nocturnal feeding
(6 – 8 h), we started the fasting period at 5– 6 AM, which represents the postprandial state. In the 14CO2 incorporation assay, 4
month-old WT with adenoviral shRNAs injection (48 h) or 5
month-old p300G422S and littermate control mice were injected ip with a dose of 2 ␮Ci/g of NaH14CO3 at 3 AM. Blood
was collected by cardiac puncture after anesthetization at 5 AM,
and livers were removed, snap-frozen in liquid nitrogen, and
stored at ⫺80°C until use. In the [2-3H]glucose incorporation
assay, 3 month-old male p300G422S and littermate control
mice were injected ip with 1 ␮Ci/g body weight of tracer at 3 AM.
After 2 hours, mice were humanely destroyed, and livers were
removed and snap-frozen in liquid nitrogen. To measure the
glucose 6-phosphate in the liver, 100 mg of hepatic tissue was
rapidly homogenized in ice-cold PBS. Following deproteinization, we used a glucose 6-phosphate assay kit to determine glucose 6-phosphate concentrations (Abcam, Inc, Cambridge,
Massachusetts). Serum lactate concentration was measured using a colorimetric lactate assay kit (Abcam). All the mice were
humanely destroyed at 5– 6 AM (postprandial state), unless otherwise specified.
Glycogen measurement
To determine 14C and [2-3H]glucose incorporation into glycogen and the glycogen content in the liver, hepatic tissues were
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p300 Maintains Glycogen Storage.
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(Abcam) and by following the procedure provided by the manufacturer.
Glycogen phosphorylase and GS
activity assays
Hepatic tissues were homogenized in
500 ␮L TES buffer (20 mM Tris, pH7.4;
1 mM EDTA; 225 mM sucrose; 2.5 mM
dithiothreitol; 0.1 mM phenylmethylsulfonylfluoride and protease inhibitor
cocktail) with a glass Dounce homogenizer as described previously (30, 31).
Samples were sonicated and centrifuged
at 13 500 rpm for 10 minutes. Protein
(100 ␮g) was used to measure glycogen
phosphorylase activity in assay buffer
containing: 50 mM potassium phosphate (pH 7.5); 10 mM MgCl2; 100 ␮M
EDTA (pH 8.0); 0.5 mM caffeine; 4 ␮M
glucose 1,6-biphosphate; 0.5 mM
NADP⫹; 1.5 U/mL glucose 6-phosphate
dehydrogenase; 1 U/mL phosphoglucomutase; 0.5 mg/mL glycogen. Blank control contained all the reagents and 100
␮g of protein except glucose 6-phosphate dehydrogenase and phosphoglucomutase. Sample absorbance at 340 nm
was measured in a spectrophotometer
after the mixture was incubated at 37°C
for 30 minutes. The amount of reduced
nicotinamide adenine dinucleotide
phosphate in the samples was calculated
by using a standard curve of known reduced nicotinamide adenine dinucleFigure 1. Important Role of CREB Coactivators in Regulating Hepatic Gluconeogenesis. A–D,
otide phosphate concentrations. GS acAdenoviral shRNAs-mediated depletion of CREB coactivators significantly lowered blood glucose
tivity was determined using a modified
levels in postprandial and/or fasted states (24 h) (n ⫽ 4 –5). The y-axis has been broken and
method of Thomas et al. (32). Hepatic
begins at 30 mg/dL. Each insert shows the knockdown effect of target protein by adenoviral
tissue was homogenized in 500 ␮L of
shRNA.
ice-cold GS assay buffer (50 mM TrisHCl, pH 7.8, 10 mM EDTA, 100 mM
KF plus proteinase inhibitor cocktail)
treated as described below. Hepatic tissue was heated at 95°C
with
a
glass
Dounce
homogenizer
prior to centrifugation at
in 30% KOH for 2 hours. Following cooling, glycogen was
10
000
⫻
g
for
20
minutes.
To
measure
the GS activity, 100 ␮g
precipitated on ice for 20 minutes with 95% ethanol. After
of total protein (5 ␮L) was added to a reaction (100 ␮L) concentrifugation, glycogen was dissolved in 300 ␮L of H2O and
taining: 50 mM Tris-HCl, pH 7.8, 10 mM EDTA, 100 mM KF,
precipitated with ethanol; this process was then repeated. The
10 mM UDP-[14C]glucose (0.1 ␮Ci/␮M), and 15 mg/mL glycoglycogen pellet was washed with 66% ethanol, recentrifuged,
gen,
in the presence or absence of 10 mM glucose 6-phosphate,
and dissolved in 300 ␮L of H2O, from which 30 ␮L was
14
3
which
allosterically activates GS. Assay conducted in the abmixed with 3 mL of liquid scintillation mixture. C and H
sence
of
glucose 6-phosphate was used to measure active GS,
were qualified using a liquid scintillation counter. To deterwhereas assay in the presence of glucose 6-phosphate was used
mine the glycogen concentration, the above purified glycogen
to measure total GS activity. Samples were incubated at 37°C,
and glycogen standards were diluted in 1 M HCl, heated at
then spotted on Whatman filter paper (Millipore Corp, Bedford,
95°C for 1 hour, and then centrifuged. The supernatants were
Massachusetts). Filter paper was, dropped immediately in 70%
diluted 25 times with H2O, and 20 ␮L of each sample was
ethanol, stirred with a stirring bar for 40 minutes, and then
used to measure glycogen content with the EnzyChrom Gluwashed twice. Filters were air dried, and radioactivity was
cose Assay Kit (BioAssay System, Hayward, California). To
counted in 3 mL liquid scintillation fluid.
correct for glucose present in the samples, we also determined
the glucose concentration in unheated samples. However,
these values turned out to be zero, indicating the purity of the
extracted glycogen. Glycogen concentrations in the primary
hepatocytes were measured by using the Glycogen Assay Kit
Immunoblot
Immunoblot was conducted as we previously described
(12, 29)
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Figure 2. The p300 Coactivator Regulates Hepatic Gluconeogenic Gene Expression in the Postprandial State. A, Depletion of p300 significantly
suppressed gluconeogenic gene expression in the liver of mice in postprandial state (left); however, depletion of CBP (right) had minimal effects on
gluconeogenic gene expression (n ⫽ 4). B, Adenoviral shRNA-mediated depletion of CRTC2 had no significant effect on the mRNA levels of
gluconeogenic genes in the postprandial state (n ⫽ 4). C and D, Hepatic protein levels of Pck1 and Fbp1(C) and serum insulin levels (D) in mice
after CBP or p300 depletion as in (A) (n ⫽ 3– 4). Each lane represents an individual mouse sample. Quantitative RT-PCR was used to measure gene
expression (normalized to 36B4 expression levels) (A and B).
Statistical analyses
Statistical significance was calculated with Student’s t test
and ANOVA test. Significance was accepted at the level of
P ⬍ .05.
Results
The distinct role of CREB coactivators in mediating
hepatic glucose production and maintaining blood
glucose levels
Previous studies have shown that the CREB coactivators are important for regulating hepatic glucose production and maintaining euglycemia (12, 33). To assess further the role of these coactivators in mediating hepatic
glucose production (HGP), we used adenoviral shRNAs
delivered through tail vein injection to deplete these coactivators in the liver (Figure 1, A–D). Depletion of p300
significantly reduced blood glucose levels in both postprandial and fasting states (Figure 1B), whereas depletion
of CBP (⬎8 h fast) and CRTC2 (⬎24 h fast) reduced
blood glucose levels only after prolonged fasting (Figure
1, C and D). Depletion of p300 resulted in the greatest
drop in blood glucose levels between 12 and 24 hours of
fasting (ad-shSCR, ⫺1.4; ad-shCBP, ⫺1.5; ad-shCRTC2,
⫺1.8; and ad-shp300, ⫺2.2 mg/dL/h). These data indicate that p300 functions differently than CBP and
CRTC2 in maintaining blood glucose levels.
Having seen that depletion of p300 in the liver led to
lower blood glucose levels and depletion of CBP and
CRTC2 had minimal effect on blood glucose levels in the
postprandial state (Figure 1, B–D), we assessed hepatic
mRNA levels of gluconeogenic genes in mice after depletion of CREB coactivators. Depletion of p300 significantly decreased mRNA levels of gluconeogenic genes; in
comparison, depletion of either CBP or CRTC2 had minimal effect on gene expression (Figure 2, A and B). In
addition, adenoviral-mediated depletion of p300 significantly decreased Pck1 protein levels without affecting serum insulin levels (Figure 2, C and D).
Role of p300 in glycogen synthesis in hepatocytes
Previous studies demonstrated that gluconeogenesis is
essential for postprandial glycogen formation (14, 17–
19). Efficient glycogen synthesis requires both glucose
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Figure 3. The p300 Coactivator Regulates Hepatic Glycogen Synthesis. A, Glycogen synthesis in primary hepatocytes is dramatically increased by
20 mM glucose and by the addition of gluconeogenic precursors (lactate and pyruvate). B, Depletion of p300 greatly decreased glycogen synthesis
wherease depletion of CBP had a mild effect on glycogen synthesis. C, Only the depletion of p300 significantly decreased hepatic glycogen
storage in mice humanely destroyed in the postprandial state (n ⫽ 7). D, Depletion of CRTC2 increased glycogen content in the liver of mice
humanely destroyed in the postprandial state (n ⫽ 4). E, Depletion of p300 significantly decreased 14C-incorporated glycogen in the 14CO2
incorporation assay in the postprandial state (n ⫽ 4). Mice were administered NaH14CO3 (2 ␮Ci/g) through ip injection 48 hours after adenoviral
shRNA injection. F, The mRNA levels of genes related to glycogen metabolism in the liver of mice with adenoviral-mediated depletion of either CBP
or p300 (n ⫽ 3– 4). The mRNA measurements were normalized to 36B4 levels. Means ⫾ SEM are shown. Asterisk (*) signifies that groups with
same treatment are significantly different (P ⬍ .05).
and gluconeogenic precursors, and this finding has been
termed the “glucose paradox” (14). We therefore conducted a glycogen synthesis assay in primary hepatocytes,
and in good agreement with previous studies, glycogen
synthesis was markedly activated only in the presence of
both high concentrations of glucose and of the gluconeogenic precursors lactate and pyruvate (Figure 3A). Thus,
p300 maintains basal gluconeogenesis (29), and depletion
of p300 led to lower blood glucose levels in the postprandial state (Figure 1B). To determine whether p300 and
other CREB coactivators have an effect on glycogen synthesis, we employed adenoviral shRNAs to deplete the
coactivators in primary hepatocytes. As shown in Figure
3B, depletion of p300 significantly decreased glycogen
formation in the presence of either glucose alone or glucose plus lactate and pyruvate as substrates, whereas CBP
depletion had a lesser effect and CRTC2 depletion had no
effect on glycogen formation. Decreased glycogen content
in hepatocytes with p300 depletion is likely due to com-
pensation by CBP, the function of which can be inhibited
by insulin-mediated phosphorylation. Furthermore, depletion of p300 significantly decreased hepatic glycogen
content in mice during the postprandial state (Figure 3C);
in contrast, depletion of either CBP or CRTC2 did not
lead to a decrease in hepatic glycogen content (Figure 3, C
and D). An early study had shown that glucose is broken
down to lactate in the liver and released into circulation
after feeding in dogs (34). One potential explanation
for glucose-stimulated glycogen synthesis is that glucose was metabolized to lactate, and lactate was then
used to synthesize glycogen through gluconeogenesis.
To unequivocally prove that p300 regulates glycogen
synthesis through gluconeogenesis, we used adenoviral
shRNA to deplete p300 and administered trace quantities
of NaH14CO3 to mice via ip injection in the postprandial
state. In this experiment, 14CO2 is incorporated into oxaloacetate through pyruvate carboxylase and subsequent
intermediates in the gluconeogenic pathway. Depletion of
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Figure 4. Up-Regulation of Gluconeogenesis Increases Glycogen Synthesis. A, The
phosphorylation status of CBP, CREB, PKC␫/␭, and Akt in the liver from fed and 16-hour fasted
mice (n ⫽ 5). B, Primary hepatocytes from CBPS436A mice produced significantly more glucose
than hepatocytes from control mice in the presence or absence of cAMP treatment. Glucose
production measurements were normalized to protein levels and the untreated control group.
The data shown here are relative to 1 (normalized data). C, Primary hepatocytes from a
CBPS436A mouse synthesized significantly more glycogen than primary hepatocytes from a
control mouse in each treatment.
14
p300 resulted in a 6-fold decrease in C-incorporated
glycogen (Figure 3E). The above data suggest that p300
plays a critical role in maintaining glyconeogenesis
through gluconeogenesis in the postprandial state.
However, mRNA levels of genes related to glycogen
synthesis were not significantly changed after p300 depletion (Figure 3F).
Increased gluconeogenesis leads to elevated
glycogen synthesis
We previously reported that CBP was phosphorylated
by insulin and metformin at S436 via atypical protein
kinase C ␫/␭ (12). This pathway was subsequently confirmed by other investigators (35). We determined hepatic
phosphorylation levels of atypical protein kinase C ␫/␭,
CBP and Akt from fed and 16-hour fasted mice and found
that they all decreased with fasting (Figure 4A). Our recent study showed that p300 maintains basal gluconeogenesis in the liver due to the lack of a corresponding
phosphorylation site found in CBP at S436, and p300
constitutively binds to the cAMP response elements
(CREs) of genes related to gluconeogenesis (29). In comparison, mutant CBP did not dissociate from the CRE site
in the liver of CBPS436A mutant mice in the fed state, and
insulin and metformin treatment had no effect on its binding (12). These data suggest that mutant CBP functions
like p300 and binds constitutively to the hepatic CRE
sites. Because the depletion of p300 decreased glycogen
synthesis in hepatocytes (Figure 3, B and C), it can be
predicted that up-regulation of gluconeogenesis in
CBPS436A mutant mice would result in increased glyco-
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gen formation. We therefore conducted glucose production and glycogen synthesis assays in primary
hepatocytes
from
CBPS436A
knock-in and littermate control
mice. Consistent with our previous
publication (36), primary hepatocytes from CBPS436A mice produced significantly more glucose
than hepatocytes from control mice
in both basal and cAMP treatment
groups (Figure 4B). We now find
that primary hepatocytes from
CBPS436A mice also synthesized
significantly more glycogen than
primary hepatocytes from control
mice in the presence of either glucose
or in the presence of glucose, lactate,
and pyruvate (Figure 4C).
Decreased gluconeogenesis in
p300G422S knock-in mice
results in the lower hepatic glycogen formation in
the postprandial state
To prove that distinct functions of p300 and CBP in
hepatic glucose production are due to the absence or presence of this phosphorylation event, we used the
p300G422S knock-in mouse model bearing the identical
phosphorylation site found in CBP at S436. We first confirmed that G422S in p300 can be phosphorylated by
insulin and metformin (29). Compared with WT littermates, mutant p300 was absent from the CRE site of
genes important for gluconeogenesis in the fed and postprandial states, and p300G422S knock-in mice also exhibited lower blood glucose levels in the postprandial
state (Figure 5A) (29). In addition, p300G422S knock-in
mice displayed hypersensitivity to insulin (Figure 5B) and
enhanced glucose tolerance (Figure 5C). Lower blood glucose levels in p300G422S knock-in mice were also associated with significantly lower protein levels of G6pc and
Pck1 (Figure 5D) and lower hepatic glycogen content in
the postprandial state (Figure 5E). We observed similar
protein levels of Gck and Glut2 in the liver of WT and
p300G422S mice (Figure 5D). Furthermore, the mRNA
levels of genes related to glycolysis and glycogen metabolism did not differ significantly between WT and
p300G422S mice (Figure 5F). Of note, p300G422S
knock-in mice had lower mRNA levels of Scd1 and Fasn,
which would decrease lipogenesis in the liver and increase
glucose tolerance (Figure 5, B, C, and F). Finally, compared with WT mice, p300G422S displayed significantly
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Figure 5. The Distinct Role of CBP and p300 in Regulating Hepatic Glucose Production due to the Presence of Insulin Phosphorylation at CBP
S436, and Which Does Not Exist in p300. A, P300G422S knock-in mice exhibited lower blood glucose levels in the postprandial state compared to
littermate control mice. B and C, Insulin tolerance test (B) and glucose tolerance test (C) in 2-hour fasted mice (n ⫽ 4 –5). D, Indicated protein
levels in the liver of p300G422S knock-in mice and littermate controls in the postprandial state (n ⫽ 4). Densitometric analysis of the protein levels
(right panel). E, Hepatic glycogen content in p300G422S knock-in mice and littermate controls in the postprandial state (n ⫽ 3– 4). F, The mRNA
levels of genes related to glucose, glycogen, and lipid metabolism in the liver of mice killed in postprandial state (n ⫽ 5). The mRNA measurements
are normalized to 36B4 levels. G and H, Hepatic glycogen contents in WT (G) and p300KI (H) mice at indicated times during the 12-hour or 24hour fasting period (n ⫽ 3– 4).
lower hepatic glycogen levels, which were quickly depleted during fasting (Figure 5, G and H).
To support the notion that decreased gluconeogenesis
resulted in the lower glycogen content in the liver of
p300G422S knock-in mice in the postprandial state (Figure 5E), we conducted glucose production assays in primary hepatocytes. Hepatocytes from p300G422S
knock-in mice produced significantly less glucose than
hepatocytes from littermate control mice in both basal
and cAMP treatment groups (Figure 6A). Insulin and metformin had a greater inhibition of cAMP-stimulated glucose production in hepatocytes from p300 knock-in mice
than in WT control mice (Figure 6, A and B). If gluconeogenesis is important for the glycogen synthesis, then decreased gluconeogenesis must result in the lower glycogen
content. We, therefore, administered trace quantities of
NaH14CO3 via ip injection to p300G422S knock-in and
WT littermate control mice in the postprandial state.
Compared with WT control mice, p300G422S knock-in
mice exhibited approximately 25⫻ less [14C]glycogen
content in the liver in the 14CO2 incorporation assay (Figure 6C). However, the protein levels of Glut2 and Gck did
not differ between p300G422S and littermate control
mice (Figure 5D), and hepatocytes from p300G422S mice
exhibited similar glucose uptake rates as hepatocytes
from WT littermate control mice (Figure 6D). In addition,
p300G422S mice displayed increased [3H]glycogen content in the liver during a [2-3H]glucose incorporation assay, even though this result did not reach statistical significance (Figure 6E). These data eliminate the possibility
that lower glycogen content in p300G422S mice is due to
decreased glucose uptake or decreased glycogen synthesis
from the direct pathway.
Next, we determined the enzymatic activity and phosphorylation status of glycogen phosphorylase. The dramatically decreased [14C]glycogen content was not due to
a change of glycogen phosphorylase activity because the
enzymatic activity and serine phosphorylation levels of
glycogen phosphorylase were not significantly different
between p300G422S and WT littermate control mice
(Figure 6, F–H). However, p300G422S mice exhibited a
significantly lower active GS and GS activity ratio (Figure
7, A–C). Intriguingly, GS was dephosphorylated in both
p300G422S and littermate control mice in the postprandial state compared with the fasting state (Figure 6G).
Moreover, p300G422S mice had similar amounts of
phospho-GS and total GS protein levels in the liver as WT
mice (Figure 6G). The above data indicate that the lower
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Figure 6. P300G422S Knock-In Mice Synthesize Less Glycogen in the Liver. A and B, Primary hepatocytes from a p300G422S knock-in mouse
produced significantly less glucose than hepatocytes from a control mouse in the presence or absence of cAMP treatment. After 4 hours of serum
starvation, cells were washed with PBS twice, after which 20 nM insulin was added 30 minutes prior to the addition of cAMP (A) and 5 mM
metformin was added 4 hours before the addition of cAMP (B). C, P300G422S knock-in mice synthesized significantly less glycogen in the 14CO2
incorporation assay (n ⫽ 4). D, [2-3H] deoxyglucose uptake in primary hepatocytes from p300KI and littermate control mice. Hepatocytes were
treated as described in “Materials and Methods”. E, P300G422S and WT control mice synthesized similar amounts of [3H]glycogen in the [23
H]glucose incorporation assay (n ⫽ 5⬃6). F, Crude extract of hepatic tissues (100 ␮g) was used to measure glycogen phosphorylase (PYG)
activity. G, Immunoblot analysis of hepatic protein levels with indicated antibodies of p300G422S knock-in mice and littermate controls in the
postprandial state. Serine phosphorylation levels were examined in immunoprecipitated glycogen phosphorylase (n ⫽ 5). H, Indicated
phosphoprotein or total protein levels were determined in the liver of WT mice killed at postprandial or fasted (24 h) state. Each lane represents an
individual mouse sample.
GS activity in the liver of p300G422S mice was not due to
changes in GS protein phosphorylation or in total GS
protein levels.
Glucose 6-phosphate is a critical allosteric activator of
GS (6, 8), and p300G422S mice had significantly lower
gluconeogenesis in the postprandial state. It is possible
that lower gluconeogenesis in the liver of p300G422S
mice would lead to lower glucose 6-phosphate levels
and subsequently affect GS activity. Indeed, compared
with WT littermate controls, p300G422S mice exhibited significantly lower hepatic glucose 6-phosphate
levels in the postprandial state (Figure 7D). The above
data suggest that lower 14C incorporation into glycogen in p300G422S knock-in mice must be due to decreased gluconeogenesis. Additionally, the reduction in
gluconeogenesis is intrinsic to the pathway as we sug-
gest because levels of serum lactate, a critical substrate
for gluconeogenesis, were higher in p300G422S
knock-in mice compared with WT littermate control
mice (Figure 7E), which suggests a reduction in lactate
utilization in the liver.
Discussion
Hepatic glycogen is important for maintaining euglycemia in early fasting but is subsequently depleted during a
prolonged fast (Figure 5G). It is generally believed that
glucose is not the primary substrate for either glyconeogenesis or lipogenesis in the liver. In contrast, gluconeogenic precursors such as lactate and pyruvate are superior
substrates for these processes (14, 15, 37, 38). It has been
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He et al
p300 Maintains Glycogen Storage.
Mol Endocrinol, August 2013, 27(8):1322–1332
Figure 7. Decreased Gluconeogenic Flux Affects Hepatic Glycogen Synthesis in p300KI Mice. A–C, GS activity in the absence or presence of 10
mM glucose 6-phosphate; active GS and GS active ratio were significantly decreased in p300G422S mice (A and B), and along with similar total GS
activity between WT and p300KI mice (C) (P ⫽ .98). D, Glucose 6-phosphate levels in the liver of p300KI and littermate control mice killed at either
postprandial or fasted (24 h) states. E, Serum lactate concentrations of p300KI and littermate control mice killed at postprandial state (n ⫽ 4). NS,
not significant. F, A proposed model for maintaining blood glucose levels by CREB coactivators through gluconeogenesis and glycogenolysis.
shown that carbon flux through the gluconeogenic pathway is essential for efficient glycogen synthesis, and the
inhibition of PCK1 enzymatic activity led to approximately 85% decrease of glycogen content in the liver (17,
18). These reports also indicate that gluconeogenesis is
still active in the postprandial states because glyconeogenesis occurs during this period. It is estimated that as much
as 50%–70% of newly synthesized glycogen is formed via
the gluconeogenic pathway (14, 21, 25). Moreover, an
unexplained observation in humans has been that gluconeogenesis is not completely suppressed even in the presence of excess insulin (39 – 42).
CBP and p300 are closely related proteins, but p300
lacks the phosphorylation site found in CBP at S436 and
cannot be phosphorylated at this site. We recently reported that p300 constitutively binds to hepatic CREs
such as found in the Ppargc1 gene promoter and maintains basal gluconeogenesis in the fed and postprandial
states (29). In the current study, we found that depletion
of hepatic p300 resulted in decreased hepatic mRNA levels of gluconeogenic genes and lower blood glucose levels
in the postprandial state. Most importantly, depletion of
p300 led to lower hepatic glycogen content and decreased
[14C]glycogen in tracer incorporation assay in the postprandial state and early fasting stages (Figure 1, Figure
2A, and Figure 3, C and E). These data suggest that p300
is an important coactivator in maintaining glyconeogenesis. Moreover, p300G422S knock-in mice exhibited
lower glycogen content and lower protein levels of gluconeogenic enzyme genes in the postprandial state (Figure 5,
D–F), and hepatocytes from p300G422S knock-in mice
produced significantly less glucose (Figure 6, A and B).
These data suggest that decreased gluconeogenesis via
introduction of an artificial phosphorylation site leads to
lower hepatic glycogen formation and causes lower blood
glucose levels in p300G422S knock-in mice in the post-
doi: 10.1210/me.2012-1413
prandial and early fasted states. This was unequivocally
substantiated by the significantly decreased [14C]glycogen in the p300G422S knock-in mice in the 14CO2 incorporation assay (Figure 6C). P300G422S knock-in mice
also had significantly lower mRNA and protein levels of
G6pc than WT control mice in the postprandial state
(Figure 5, D and F), which would, if anything, tend to
divert the reduced gluconeogenic flux toward glycogen
synthesis. These data suggest that constitutive binding of
p300 to gluconeogenic enzyme gene promoters is also
necessary for maintaining hepatic glycogen stores. On the
other hand, mice containing CBPS436A, which functions
like p300, displayed increased gluconeogenesis and glycogen synthesis in hepatocytes (Figure 4, B and C) (12,
36). Thus, the activation of gluconeogenic pathway by
constitutive binding of p300 to the CREB coactivator
complex maintains glyconeogenesis and sustains euglycemia through the activation of basal gluconeogenesis in the
postprandial and early fasting states (Figure 7F).
Given that diabetic patients have increased hepatic gluconeogenesis (42, 43), one might predict that hepatic glycogen storage would be increased. In fact, however, previous studies have shown that diabetic patients have
lower hepatic glycogen content due to decreased GS
and/or increased glycogen phosphorylase activity (42,
43). This discrepancy might be explained by an increase in
glucose 6-phosphatase in diabetic patients (44 – 46),
which would increase the conversion of glucose 6-phosphate to glucose rather than being used for glycogen synthesis. Our mouse models of hepatic p300 depletion (Figures 2 and 3) and p300G422S mutation (Figures 5–7)
demonstrate that p300 is critical for basal gluconeogenesis and glycogen synthesis and that p300 is unique among
the CREB coactivators in having this function. In comparison, neither depletion of CBP nor CRTC2 resulted in
the reduction of postprandial hepatic glycogen levels (Figure 3, C and D). Because total protein and phosphorylation levels of glycogen phosphorylase and synthase were
normal in p300G422S mutant mice, the enzymatic activity of glycogen phosphorylase did not differ between WT
and mutant mice (Figure 6, F and G). In contrast, we
demonstrate that the lower glycogen content in mutant
mice was due to the decreased GS activity resulting from
decreased glucose 6-phosphate levels (Figure 7). Glucose
6-phosphate can be formed from glucose after its uptake
and phosphorylation by glucokinase or be derived from
gluconeogenesis. Given that glucose 6-phosphatase
mRNA and protein levels are reduced, glucose uptake and
protein levels of Glut2 and Gck are normal, and glycogen
synthesis from direct pathways is unaffected in hepatocytes from p300G422S mice; the lower glucose 6-phosphate levels in the liver of p300G422S mice in the post-
mend.endojournals.org
1331
prandial state must be due to a marked decrease in hepatic
gluconeogenesis. Furthermore, p300 maintains constitutive activation of gluconeogenesis even in the postprandial state, which is due to the fact that p300 activity is not
regulated by insulin (29). The latter finding may explain
rodent and human data, which suggest that gluconeogenesis is unable to be completely inhibited even when high
serum insulin levels are achieved in insulin-sensitive normal animals and human subjects.
Acknowledgments
Address all correspondence and requests for reprints to: Ling
He, Division of Metabolism, 600 North Wolfe Street,
CMSC10 –113, Johns Hopkins University School of Medicine,
Baltimore, Maryland 21287. E-mail: [email protected].
This work was supported by grants from the National
Institute of Diabetes and Digestive and Kidney Diseases,
R00DK085142 (to L.H.) and R01DK063349 (to F.E.W.); and
by the Baltimore Diabetes Research and Training Center,
P60DK079637 (to F.E.W.).
Disclosure Summary: The authors have nothing to disclose.
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CBP, CREB-binding protein; CRE, cAMP response element; CREB,
CRE-binding protein; GS, glycogen synthase; shRNA, short hairpin
RNA; WT, wild type.