Am J Physiol Endocrinol Metab 291: E358 –E364, 2006;
doi:10.1152/ajpendo.00027.2006.
Deficiency of carbohydrate-activated transcription factor ChREBP prevents
obesity and improves plasma glucose control in leptin-deficient (ob/ob) mice
Katsumi Iizuka,1 Bonnie Miller,2 and Kosaku Uyeda1,3
Departments of 1Biochemistry and 2Internal Medicine, University of Texas
Southwestern Medical School; and 3Veterans Affairs Medical Center, Dallas, Texas
Submitted 19 January 2006; accepted in final form 13 March 2006
may be the single most significant cause of the
current epidemic of obesity and obesity-associated diseases
occurring in the United States. Mechanisms that result in the
complex of metabolic derangements that specifically increase
risk for type 2 diabetes, heart disease, nonalcoholic fatty liver
disease, and other obesity-associated disorders are not completely understood, however, impeding development of effective strategies to combat the pathological consequences of
caloric excess. The tendency of obesity to disrupt normal
mechanisms of appetite control exacerbates the problem (30).
When carbohydrate intake exceeds short-term requirements
for energy or repleting glycogen, metabolizable sugars are
converted to fatty acids for long-term storage as fat by the
combined activities of the glycolytic and lipogenic pathways in
liver. Eating a diet that is high in carbohydrates induces gene
transcription of over a dozen enzymes in liver that are involved
in the metabolic conversion of carbohydrate to storage fat
(8, 12, 25, 26). Insulin, which is secreted by the pancreas in
response to elevated blood glucose levels when carbohydrates are eaten, is well known to promote lipogenesis and
to increase transcription of many lipogenic enzyme genes.
However, nutrients, especially carbohydrates, also play an
important role in the transcriptional regulation of many of
the same lipogenic genes by mechanisms that are independent of hormone signaling.
The mechanism by which excess carbohydrate activates a
glucose-signaling pathway to induce transcription of lipogenic
enzyme genes independently of insulin became clearer with the
discovery of the transcription factor carbohydrate response
element-binding protein (ChREBP) (29). When glucose availability is low, a phosphorylated, inactive pool of ChREBP is
maintained in the hepatocyte cytosol (16). When glucose
availability increases, glucose metabolism through the pentose
shunt pathway increases, resulting in an increased concentration of the metabolite xyulose 5-phosphate. Xyulose 5-phosphate activates a specific protein phosphatase that dephosphorylates ChREBP (15), which then is translocated to the nucleus
where it binds to carbohydrate response elements in the promoters of lipogenic enzyme genes (14) and the gene encoding
the glycolytic enzyme liver pyruvate kinase [LPK (29)] to
coordinately activate their transcription.
In mice with a targeted disruption of the ChREBP gene
(ChREBP⫺/⫺), de novo fatty acid synthesis and overall adiposity are decreased compared with wild-type (WT) mice (13).
ChREBP⫺/⫺ mice do, however, store modest amounts of fat
obtained directly from the diet. Liver triglyceride storage is
reduced significantly in ChREBP⫺/⫺ mice ad libitum fed a
high-carbohydrate diet compared with similarly fed WT mice
(13). To investigate the effect of ChREBP deficiency on
development of obesity and metabolic dysregulation resulting
from caloric excess in the studies reported here, ChREBP⫺/⫺
and ob/ob mice were intercrossed to produce doubly deficient
ob/ob-ChREBP⫺/⫺ mice. The obesity gene (ob) encodes
leptin, an adipocyte hormone that regulates the neuroendocrine response to nutrients (4, 7). As the result of a mutation
in the ob gene, ob/ob mice do not express leptin, their food
intake is increased, and thermogenesis is decreased (4).
ob/ob mice become obese and insulin resistant and develop
fatty livers. Hyperinsulinemia in ob/ob mice occurs prior to
marked hyperphagia or obesity (1, 11) and may result from
a direct effect of leptin on pancreatic ␤-cells to suppress
insulin secretion (17).
Address for reprint requests and other correspondence: K. Uyeda, Dept. of
Biochemistry, Univ. of Texas Southwestern Medical School, 4500 S. Lancaster Rd., Dallas, TX 75216 (e-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
carbohydrate response element-binding protein
OVEREATING
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Iizuka, Katsumi, Bonnie Miller, and Kosaku Uyeda. Deficiency
of carbohydrate-activated transcription factor ChREBP prevents obesity and improves plasma glucose control in leptin-deficient (ob/ob)
mice. Am J Physiol Endocrinol Metab 291: E358 –E364, 2006;
doi:10.1152/ajpendo.00027.2006.—The transcription factor carbohydrate response element-binding protein (ChREBP) mediates insulinindependent, glucose-stimulated gene expression of multiple liver
enzymes responsible for converting excess carbohydrate to fatty acids
for long-term storage. To investigate ChREBP’s role in the development of obesity and obesity-associated metabolic dysregulation,
ChREBP-deficient mice were intercrossed with ob/ob mice. As a
result of deficient leptin expression, ob/ob mice overeat, become
obese and resistant to insulin, and display marked elevations in
hepatic lipogenesis, gluconeogenesis, and plasma glucose and triglycerides. mRNA expression of all hepatic lipogenic enzymes was
significantly lower in ob/ob-ChREBP⫺/⫺ than in ob/ob mice, resulting in decreased hepatic fatty acid synthesis and normalization of
plasma free fatty acid and triglyceride levels. Overall weight gain in
addition to adiposity was reduced in the doubly deficient mice. The
former was largely attributable to decreased food intake and may
result from decreased hypothalamic expression of the appetite-stimulating neuropeptide agouti-related protein. mRNA expression and
activity of gluconeogenic enzymes also was lower in the doubly
deficient mice, contributing to significantly lower blood glucose
levels. The results of this study suggest that inactivation of ChREBP
expression not only reduces fat synthesis and obesity in ob/ob mice
but also results in improved glucose tolerance and appetite control.
CHREBP DEFICIENCY PREVENTS OBESITY IN
RESEARCH DESIGN AND METHODS
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Quantitative real-time RT-PCR. Total RNA was extracted using
TRIzol reagent (Invitrogen, Carlsbad, CA), and cDNA was synthesized using the SuperScript II System (Invitrogen). Quantitative PCR
was performed using the I-cycler system (Bio-Rad). Primer pairs for
liver enzymes were those described previously (13). Primer pairs for
neuropeptide mRNA quantitation were as follows: proopiomelanocortin (POMC) F 5⬘-cctggcaacggagatgaac, R 5⬘-ccacaccgcctcttcctc; agouti-related protein (AgRP) F 5⬘-tggcggaggtgctagatc, R 5⬘-cattgaagaagcagcggcagtag; cocaine- and amphetamine-regulated transcript
(CART) F 5⬘-ctactctgccgtggatgat, R 5⬘-tcttgagcttcttcaggacttc; and
neuropeptide Y (NPY) F 5⬘-ctcgtgtgtttgggcattc, R 5⬘-gattgatgtagtgtcgcagag.
Experimental mRNA levels were normalized to cyclophilin mRNA
in the same sample, and the comparative cycle threshold method was
used to compare the normalized mRNA values between animals.
Insulin receptor substrate (IRS)-2/IRS-1 mRNA ratios were calculated
without correction for any minor differences in SYBR Green binding
to the respective PCR products. Taq polymerase was purchased from
Promega (Madison, WI).
Liver metabolites and enzyme activities. Animals were killed by
cervical dislocation between 8:00 and 9:00 AM; their livers were
removed immediately, freeze-clamped between aluminum blocks
cooled in liquid nitrogen, and ground to powder while frozen. Perchloric acid extracts of a portion of the frozen liver powder were
prepared and neutralized with KHCO3, and the resulting supernatant
solutions were used for metabolite measurements in standard assays
(2a). By use of enzymes purchased from Roche Diagnostics (Indianapolis, IN), glucose and glucose 6-phosphate were determined using
hexokinase and glucose-6-phosphate dehydrogenase, pyruvate and
phosphoenolpyruvate were determined using lactate dehydrogenase
and pyruvate kinase, and lactate and malate were determined using
lactate dehydrogenase and malate dehydrogenase, respectively. Fructose-2,6-bisphosphate was determined using pyrophosphate:fructose6-phosphate phosphotransferase (Sigma). Standard assays were used
to measure hepatic enzyme activities in additional portions of the
same liver samples (2a).
Insulin resistance test. Fed mice were injected intraperitoneally
with 1 U/kg body wt human insulin (Sigma), and plasma glucose
concentrations were measured using AACU-Chek Active (Roche
Diagnostics).
RESULTS
Phenotypic characteristics of ob/ob-ChREBP⫺/⫺ mice.
Gross phenotypic characteristics of WT, ChREBP⫺/⫺, ob/ob,
and ob/ob-ChREBP⫺/⫺ mice are compared in table 1. Weight
gain was significantly reduced in ob/ob-ChREBP⫺/⫺ mice
compared with ob/ob mice. The body weight of ob/ob-
Table 1. Phenotypic characteristics of WT, ChREBP⫺/⫺, ob/ob, and ob/ob-ChREBP⫺/⫺ mice at 6 wk of age
Body weight, g
Liver weight, g
Epididymal fat, weight, %body wt
Brown fat weight, % body wt
Plasma glucose, mg/dl
Plasma insulin, pmol/l
Plasma FFA, mM
Plasma triglyceride, mg/dl
Liver glycogen, ␮mol/g
Liver triglyceride, mg/g
WT
ChREBP⫺/⫺
ob/ob
ob/ob⫺ChREBP⫺/⫺
18⫾0.7
0.84⫾0.07
1.1⫾0.1
0.50⫾0.04
161⫾8
60⫾9
0.64⫾0.06
136⫾24
145⫾10
16.2⫾2.5
19.8⫾1.0
1.2⫾0.08
0.8⫾0.1*
0.49⫾0.03
208⫾10*
78⫾9
0.63⫾0.05
131⫾12
237⫾8.0*
12.1⫾1.9*
31.2⫾1.3
2.3⫾0.09
5.8⫾0.5
1.66⫾0.15
401⫾41
792⫾75
0.78⫾0.06
297⫾50
183⫾8.0
95.5⫾20
20.1⫾0.6†
3.8⫾0.02†
1.8⫾0.2†
0.89⫾0.03†
287⫾29†
738⫾76
0.46⫾0.06†
131⫾17†
441⫾38†
12.8⫾2.4†
Values presented are means ⫾ SE; n ⫽ at least 6 mice. WT, wild type; ChREBP⫺/⫺, carbohydrate response element-binding protein deficient; ob/ob, leptin
deficient; ob/ob-ChREBP⫺/⫺, doubly deficient; FFA, free fatty acids. Tissue and plasma determinations were made in mice killed between 8 AM and 9 AM using
assays described in RESEARCH DESIGN AND METHODS. *P ⬍ 0.05 vs. WT mice; †P ⬍ 0.05 vs. ob/ob mice.
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Materials. Standard laboratory chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless specified otherwise.
Animals. All studies were approved by the University of Texas
Southwestern Medical Center Institutional Animal Care and Use
Committee. All mice used in these studies were male. The generation
of ChREBP⫺/⫺ mice was described previously (13). Mice were
maintained on a 12:12-h light-dark cycle and had ad libitum access to
standard laboratory chow (Harlan-Teklad Mouse/Rat Diet 7002; Harlan-Teklad Premier Laboratory Diets). Lepob/⫹ mice (C57BL6/6J;
Jackson Laboratories, Bar Harbor, ME) and ChREBP⫺/⫺ mice were
intercrossed to produce double heterozygous (ChREBP⫺/⫹-ob/⫹)
mice, which were intercrossed to produce double homozygous (ob/
ob-ChREBP⫺/⫺) mice. Because of the low fertility of ob/ob mice,
Lepob/⫹ mice were intercrossed to produce ob/ob offspring for study.
ChREBP and Lep genotyping were done by PCR. Genotyping for the
ChREBP mutation was described previously (13). The genotyping
strategy for the intercrossed mice was based on the fact that the ob
mutation generates a DdeI restriction site. A 160-bp region spanning
the site of the ob mutation was amplified by polymerase chain reaction
using oligonucleotides 5⬘-TGTCCAAGATGGACCAGACTC-3⬘ and
5⬘-ACTGGTCTGAGGCAGGGAGCA-3⬘, digested with DdeI, and
the products resolved on a 3% agarose gels.
Blood and tissue sampling. Plasma insulin, free fatty acids, and
triglyceride concentrations were measured using Grazyme Insulin
EIA and NEFA-C Test kits (Wako Pure Chemical, Osaka, Japan) and
Triglyceride Reagent (Sigma-Aldrich). Liver triglyceride and glycogen contents were determined as described previously (13).
Hepatocyte isolation. Hepatocytes were isolated using a collagenase perfusion method with some modifications to our previously
described procedure (14). Perfusate solutions were maintained at
32°C to decrease hepatic O2 consumption during perfusion, and the
bilateral renal artery and vein and celiac artery were ligated to
maximize perfusion of the liver, following the suggestions of Y.
Tochino (Dept. of Internal Medicine and Molecular Science, Osaka
University).
De novo lipogenesis in hepatocytes. Hepatocytes were allowed to
attach to culture wells in DMEM culture medium containing 100 nM
dexamethasone, 10 nM insulin, and 100 nM 3,3⬘,5-triiodothyronine
for 3 h. Nonadherent cells were removed, and hepatocyte monolayers
were incubated an additional 3 h in the same medium containing
[U-14C]glucose (Amersham, Piscataway, NJ) and a total glucose
concentration of 5.5 or 25 mM. At the end of the incubation period,
the cells were harvested, total lipids were extracted by the Bligh-Dyer
method (3), and incorporated 14C radioactivity was measured by
liquid scintillation counting. The water exchange ratio was measured
using 3H-labeled water (Amersham) and was ⬃60%.
OB/OB
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CHREBP DEFICIENCY PREVENTS OBESITY IN
Fig. 1. Insulin resistance in wild-type (WT), carbohydrate response elementbinding protein-deficient (ChREBP⫺/⫺), leptin-deficient (ob/ob), and doubly
deficient ob/ob-ChREBP⫺/⫺ mice. Plasma glucose levels in fed mice were
determined after ip administration of 1 U/kg body wt insulin. Values presented
are means ⫾ SD from 6 mice per group. *1P ⬍ 0.05 vs. WT; *2P ⬍ 0.05 vs.
ob/ob.
AJP-Endocrinol Metab • VOL
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Table 2. Relative liver enzyme mRNA levels in WT,
ChREBP⫺/⫺, ob/ob, and ob/ob-ChREBP⫺/⫺ mice
fed a standard chow diet
GK
PFK
PK
G-6-Pase
PEPCK
FAS
ACC
SCD
PPAR␣
PPAR␦
PPAR␥
SREBP-1
PGC-1␣
ChREBP
IRS-2/IRS-1
ChREBP⫺/⫺
ob/ob
ob/ob-ChREBP⫺/⫺
0.71⫾0.08
1.57⫾0.15
0.43⫾0.03*
0.43⫾0.03*
0.75⫾0.09
0.67⫾0.04*
0.86⫾0.04
0.49⫾0.08*
0.78⫾0.18
1.53⫾0.16
1.20⫾0.24
1.10⫾0.03
0.77⫾0.14
0
1.15⫾0.38
5.12⫾0.86
6.64⫾0.21
12.30⫾1.20
1.95⫾0.06
0.72⫾0.04
10.8⫾0.70
4.15⫾0.23
12.3⫾0.19
1.63⫾0.09
5.88⫾0.48
6.55⫾0.55
5.84⫾0.24
0.8⫾0.10
2.11⫾0.20*
0.54⫾0.14*
6.08⫾0.74
8.49⫾0.64
2.37⫾0.28†
0.28⫾0.04†
0.34⫾0.05†
1.66⫾0.44†
1.15⫾0.24†
3.38⫾0.48†
1.15⫾0.19
5.32⫾0.49
5.01⫾0.51
3.55⫾0.35†
0.40⫾0.03†
0
0.99⫾0.30
Data are presented as means ⫾ SD; n ⫽ 4 – 6 mice per group. GK,
glucokinase; PFK, phosphofructokinase; PK, pyruvate kinase; G-6-Pase, glucose-6-phosphatase; PEPCK, phosphoenolpyruvate carboxykinase; ACC,
acetyl-CoA carboxylase; FAS, fatty acid synthase; SCD, stearoyl-CoA dehydrogenase; PPAR, peroxisome proliferator-activated receptor; SREBP-1, sterol
regulatory element-binding protein-1; PGC-1␣, PPAR␥ coactivator-1␣; IRS,
insulin receptor substrate. WT, ChREBP⫺/⫺, ob/ob, and ob/ob-ChREBP⫺/⫺
male mice were housed with ad libitum access to standard rodent chow and, at
6 wk of age, were killed and their livers freeze-clamped between 8 AM and 9
AM. Indicated mRNA levels were measured by real-time RT-PCR as described in RESEARCH DESIGN AND METHODS and normalized to cyclophilin
mRNA determined in the same sample. The comparative cycle threshold
method was used to determine expression levels of each mRNA species
relative to those in WT mice, which are arbitrarily assigned a value of 1. *P
⬍ 0.05 vs. WT; †P ⬍ 0.05 vs. ob/ob.
were determined (Table 2). In liver from ob/ob mice fed a
standard diet, all glycolytic and lipogenic enzyme mRNA
levels measured were 4 –15 times higher than those in WT
mice. In ob/ob-ChREBP⫺/⫺ mouse liver, mRNA levels for
lipogenic enzymes, i.e., acetyl-CoA carboxylase (ACC), fatty
acid synthase (FAS), and stearoyl-CoA dehydrogenase (SCD),
were significantly lower than in ob/ob mice and were comparable with those in ChREBP⫺/⫺ mice. SREBP-1c mRNA,
however, was significantly higher in both ob/ob and ob/obChREBP⫺/⫺ mice than in either WT or ChREBP⫺/⫺ mice.
mRNA expression of peroxisome-proliferator-activated receptor-␥ (PPAR␥), a minor PPAR isoform in liver implicated in
fatty liver development in ob/ob mice (19), as well as that of
another minor isoform, PPAR␦, was high in liver of ob/ob
mice. In ob/ob-ChREBP⫺/⫺ mice, mRNA expression of both
minor isoforms was slightly lower than in ob/ob mice but still
considerably higher than in WT mice. mRNA expression for
the predominant liver isoform, PPAR␣, which regulates gene
expression of enzymes involved in fatty acid oxidation (2), was
slightly higher in ob/ob than in WT mice and slightly lower in
ob/ob-ChREBP⫺/⫺ mice. These results suggest that transcriptional activation by ChREBP is critical for induction of lipogenic enzyme gene expression in ob/ob mice. Of the glycolytic
enzyme mRNAs examined, only that for LPK was lower in
ob/ob-ChREBP⫺/⫺ than in ob/ob mice, consistent with identification of ChREBP as the major regulator of LPK gene
transcription.
Glucose-6-phosphatase (G-6-Pase) catalyzes the final step in
glycogenolysis and gluconeogenesis and is needed for glucose
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ChREBP⫺/⫺ animals at 6 wk of age was just 60% that of
age-matched ob/ob mice and not different from ChREBP⫺/⫺
mice. Liver weights of ob/ob-ChREBP⫺/⫺ mice, however,
were significantly higher even than those of ob/ob mice, but
hepatomegaly in ob/ob-ChREBP⫺/⫺ mice resulted from high
liver glycogen content and not fat storage. The glycogen
concentration in livers from ob/ob-ChREBP⫺/⫺ mice was 2.4and 3.0-fold that in ob/ob and WT mice, respectively. Liver
triglyceride levels in ob/ob-ChREBP⫺/⫺ mice in contrast were
not different from those in WT mice. Plasma triglyceride and
free fatty acid levels in ob/ob-ChREBP⫺/⫺ mice were similar
to those in WT and ChREBP⫺/⫺ mice, and epididymal and
brown adipose tissue weights in ob/ob-ChREBP⫺/⫺ mice were
significantly lower than those in ob/ob animals.
Plasma glucose levels in ob/ob-ChREBP⫺/⫺ mice also were
lower than those in ob/ob mice, by ⬃30% and only somewhat
higher than those in ChREBP⫺/⫺ mice. Nonetheless, plasma
insulin levels were not significantly different between ob/ob
and ob/ob-ChREBP⫺/⫺ mice and were over 10-fold higher
than those in WT mice. Systemic insulin resistance in ob/obChREBP⫺/⫺ mice was comparable to that of ob/ob mice (Fig.
1), suggesting that lower plasma glucose levels in the doubly
deficient mice do not result from an increase in insulinmediated glucose disposal. As observed previously (13),
ChREBP⫺/⫺ mice are modestly insulin resistant (Fig. 1),
consistent with small but significant increases in their plasma
insulin and glucose levels compared with WT littermates
(Table 1).
Liver enzyme and transcription factor expression. Insulin
promotes lipogenesis by activating the transcription factor
sterol response element-binding protein (SREBP)-1c (6, 9, 10,
18). However, ChREBP is required for maximal induction of
lipogenic enzyme gene expression in liver, as well as that of
LPK (13). In ob/ob mice, the level of ChREBP mRNA is twice
that in WT animals (Table 2), consistent with their elevated
blood glucose. To investigate whether ChREBP deficiency in
ob/ob-ChREBP⫺/⫺ mice results in decreased gene expression
of key glycolytic and lipogenic enzymes, levels of mRNA
encoding these enzymes in livers of the different mouse strains
OB/OB
CHREBP DEFICIENCY PREVENTS OBESITY IN
Table 3. Liver enzyme activities in WT, ChREBP⫺/⫺, ob/ob,
and ob/ob-ChREBP⫺/⫺ mice fed a standard chow diet
WT
ob/ob
ob/ob
ChREBP⫺/⫺
6.7⫾1.4
5.5⫾0.3
125⫾1.9
80.6⫾13.9
21.5⫾0.8
9.8⫾0.2
4.1⫾0.5
14.2⫾0.9
5.7⫾1.1
7.1⫾0.5
54⫾0.6*
23.3⫾3.8*
15.2⫾3.1
4.0⫾0.7*
1.9⫾0.2*
9.5⫾1.1
20.0⫾0.5
10.7⫾1.3
134⫾3.6
164⫾26.4
39.3⫾2.9
24.4⫾1.5
25⫾2.3
76.8⫾1.5
17.5⫾1.2
12.5⫾3.0
44⫾0.7†
9.5⫾1.2†
12.0⫾0.2†
3.3⫾0.5†
5.2⫾0.7†
17.5⫾1.5†
9.4⫾1.5
39.2⫾0.6
2.3⫾0.1
3.2⫾0.20*
15.6⫾0.5*
1.7⫾0.1*
60.6⫾6.2
145⫾2.7
1.4⫾0.1
15.1⫾2.0†
46.5⫾1.6†
1.2⫾0.1
Data are presented as means ⫾ SD; n ⫽ 4 – 6 mice per group. ME, malic
enzyme. Liver enzyme activities were determined as described in RESEARCH
DESIGN AND METHODS, using a portion of liver samples described in Table 2.
Individual enzyme activities are given as mU/mg liver protein. GK/G-6-Pase
ratios in livers from WT, ChREBP⫺/⫺, ob/ob, and ob/ob-ChREBP⫺/⫺ mice
were 0.08, 0.24, 0.12, and 1.84, respectively. Fatty acid synthesis and ␤-oxidation rates were determined in isolated hepatocytes and are given as nmol
glucose䡠h⫺1䡠mg liver protein⫺1 incorporated into lipid and nmol
palmitate䡠h⫺1䡠mg liver protein⫺1 oxidized, respectively. *P ⬍ 0.05 vs. WT;
†P ⬍ 0.05 vs. ob/ob.
release from liver. G-6-Pase mRNA levels were elevated in
ob/ob mice compared with WT and ChREBP⫺/⫺ mice (Table
2), consistent with high hepatic glucose output in ob/ob mice
(5). mRNA expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), however, was modestly reduced compared with WT mice. In ob/ob-ChREBP⫺/⫺
mice, PEPCK mRNA was even lower than in ob/ob mice, and
G-6-Pase mRNA levels also were lower than those in WT or
ChREBP⫺/⫺ mice, suggesting a decrease in hepatic gluconeogenesis and glucose output in the doubly deficient mice.
mRNA expression of PPAR␥ coactivator-1␣ (PGC-1␣), a
coactivator of PEPCK gene transcription, paralleled that of
PEPCK mRNA in all strains examined.
The insulin signaling protein IRS-2 is implicated in insulin
suppression of hepatic glucose output (21, 27). In ob/ob mice,
IRS-2 mRNA levels tended to be lower, whereas IRS-1 mRNA
levels tended to be higher, resulting in a significant decrease in
IRS-2/IRS-1 mRNA ratio (Table 2). ob/ob-ChREBP⫺/⫺ mice
expressed WT levels of both mRNAs.
To investigate the extent to which observed differences in
glycolytic, gluconeogenic, and lipogenic enzyme mRNA expression are reflected by differences in enzymatic activities in
MICE
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these mouse strains, total activity of selected enzymes (Table
3) and intermediate metabolite content (Table 4) in liver were
determined. The latter measurements are particularly important
for determining the net in vivo activity of liver glycolysis
because of the extensive regulation of many glycolytic enzymes by allosteric effectors and the concomitant activity of
the opposing gluconeogenic pathway.
Glucose phosphorylation, primarily by glucokinase (GK) in
liver, is required for glucose entry into the glycolytic pathway
or for storage as glycogen. GK activity in both ob/obChREBP⫺/⫺ and ob/ob mice was nearly threefold that in WT
mice and was consistent with their higher liver GK mRNA
levels. In ob/ob mice, however, activity of the opposing enzyme G-6-Pase also was elevated more than twofold compared
with the WT value. Consistent with increased activities of both
enzymes, liver glucose 6-phosphate content in ob/ob mice was
only modestly elevated (Table 4). In contrast, in livers of
ob/ob-ChREBP⫺/⫺ mice, G-6-Pase activity was only one-tenth
the WT value. Consistent with a low G-6-Pase/GK ratio,
glucose 6-phosphate content in ob/ob-ChREBP⫺/⫺ mouse liver
was nearly 10-fold that in WT mice. The low G-6-Pase/GK
ratio is likely to contribute to elevated liver glycogen storage in
ob/ob-ChREBP⫺/⫺ mice.
Pyruvate, lactate, and malate levels are significantly altered
in ChREBP⫺/⫺ mice. The redox state in liver of ChREBP⫺/⫺
mice is markedly reduced on the basis of the calculation of
NAD/NADH and NADP/NADPH couples using these values.
We are currently investigating the reason for this unusual
change in the redox potential in these mice.
As predicted by decreased mRNA expression of lipogenic
enzymes in ob/ob-ChREBP⫺/⫺ mice, enzymatic activities of
the lipogenic enzymes ACC and FAS, and that of malic
enzyme, which provides NADPH for lipogenesis, were all
decreased significantly in ob/ob-ChREBP⫺/⫺ compared with
ob/ob mouse liver (Table 3). Consistent with the 75– 85%
decreases measured in the individual enzyme activities, fatty
acid synthesis from glucose in hepatocytes isolated from ob/
ob-ChREBP⫺/⫺ mice was reduced by over 70% compared
with that in hepatocytes from ob/ob animals under conditions
of both low and high glucose concentrations (Table 3). Fatty
acid ␤-oxidation in hepatocytes isolated from ob/obChREBP⫺/⫺ mice, however, was even lower than in hepatocytes isolated from ob/ob mice (Table 3). Thus decreased fatty
acid synthesis in liver of ob/ob-ChREBP⫺/⫺ mice, and not an
increase in fatty acid ␤-oxidation, appears to account for their
lower levels of liver and plasma triglycerides.
Table 4. Intermediate metabolites in liver of WT, ChREBP⫺/⫺, ob/ob, and ob/ob-ChREBP⫺/⫺ mice fed a standard chow diet
Glucose
G-6-P
F-2,6-P2
PEP
Pyruvate
Lactate
Malate
WT
ChREBP⫺/⫺
ob/ob
ob/ob⫺ ChREBP⫺/⫺
4.9⫾0.3
0.14⫾0.02
0.0077⫾0.0008
0.12⫾0.01
0.20⫾0.01
1.41⫾0.31
0.64⫾0.04
7.1⫾0.8*
0.33⫾0.06*
0.0102⫾0.0007
0.22⫾0.01*
0.13⫾0.02*
1.75⫾0.17
1.16⫾0.01*
7.0⫾0.9
0.22⫾0.02
0.0188⫾0.002
0.22⫾0.03
0.39⫾0.03
1.68⫾0.17
1.23⫾0.19
4.8⫾0.1†
1.03⫾0.07†
0.016⫾0.0006
0.29⫾0.04
0.19⫾0.01†
2.32⫾0.22
1.14⫾0.23
Data are presented as means ⫾ SD; n ⫽ 4 – 6 mice per group. G-6-P, glucose 6-phosphate; F-2,6-P2, fructose-2,6-bisphosphate; PEP, phosphoenolpyruvate.
Liver metabolites (␮mol/g liver) were quantified by standard assays as described in RESEARCH DESIGN AND METHODS, using a portion of liver samples described
in Table 2. *P ⬍ 0.05 vs. WT; †P ⬍ 0.05 vs. ob/ob.
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GK
PFK
PK
G-6-Pase
PEPCK
ACC
FAS
ME
Fatty acid synthesis
5.5 mM Glc
25 mM Glc
Oxidation
ChREBP⫺/⫺
OB/OB
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CHREBP DEFICIENCY PREVENTS OBESITY IN
Fig. 2. Weight gain in WT, ChREBP⫺/⫺, ob/ob, and ob/ob-ChREBP⫺/⫺ mice.
Body weights of ad libitum-fed mice beginning at 4 wk of age were measured
weekly at 11 AM. Values presented are means ⫾ SD from 6 mice per group.
*P ⬍ 0.05 vs. ob/ob mice from 4 to 15 wk.
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Table 5. Effect of calorie restriction on body weight, liver
weight, and liver glycogen and triglycerides in
ob/ob-ChREBP⫺/⫺ mice
Body weight, g
Liver, % body
weight
Liver triglyceride,
mg/g
Liver glycogen,
␮mol/g
WT
ChREBP⫺/⫺
ob/ob
ob/ob-ChREBP⫺/⫺
24.9⫾0.5
24.1⫾0.8
4.4⫾0.3
4.4⫾0.2
15.8⫾1.8
12.9⫾0.2
142⫾14
132⫾10
24.4⫾1.2
23.8⫾0.3
5.7⫾0.3
4.7⫾0.2*
17.3⫾2.8
19.8⫾1.3
245⫾9
182⫾23*
51.8⫾0.8
41.7⫾1.9*
7.3⫾0.4
4.2⫾0.1*
110⫾5.5
52.2⫾9.4*
295⫾12
100⫾1*
35.5⫾0.9
30.7⫾1.9*
17.5⫾0.8
15.0⫾0.9
17.8⫾2.7
18.1⫾0.5
455⫾9
368⫾25*
Data are presented as means ⫾ SD. Male mice at 9 wk of age (n ⫽ 5 per
group) were killed and liver composition determined (1st value in each
category) or were fed 4.4 g of standard chow diet daily for an additional 20
days before being killed (2nd value in each category). *P ⬍ 0.05 vs. standard
chow diet.
ob/ob mice, consistent with their high food intake, NPY and
AgRP mRNAs were much higher than in WT mice, whereas
CART and POMC mRNA levels were much lower. In ob/obChREBP⫺/⫺ mice, mRNA expression of anorexic neuropeptides did not differ from that in ob/ob mice. However, NPY
mRNA was somewhat higher, whereas mRNA for AgRP was
lower, ⬃30% less, suggesting that this hormone might be
responsible for reduced food intake in ob/ob-ChREBP⫺/⫺
mice. Consistent with this possibility, intracerebroventricular
injection of AgRP to ob/ob-ChREBP⫺/⫺ mice increased their
food intake to levels comparable to those of ob/ob mice (data
not shown).
DISCUSSION
Previously, we demonstrated (13) that ChREBP⫺/⫺ mice
exhibit phenotypic effects that indicate that ChREBP contributes to both basal and carbohydrate-induced expression of all
Fig. 3. Orexic and anorexic hormone mRNA levels in hypothalamus of WT,
ChREBP⫺/⫺, ob/ob, and ob/ob-ChREBP⫺/⫺ mice. NPY, neuropeptide Y;
AgRP, agouti-related protein; CART, cocaine- and amphetamine-regulated
transcript; POMC, proopiomelanocortin. Hypothalami from ad libitum-fed
mice were removed between 8 AM and 9 AM. Neuropeptide mRNA levels
were measured by real-time RT-PCR and normalized to cyclophilin mRNA
measured in the same sample. The normalized WT mRNA level for each
neuropeptide was assigned a value of 1, and neuropeptide mRNA levels in
hypothalamus of the experimental groups relative to WT were determined
using the comparative cycle threshold method. Values presented are assay
means ⫾ SD. *P ⬍ 0.05 ob/ob-ChREBP⫺/⫺ vs. ob/ob.
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Food consumption. To investigate whether a decrease in
food consumption might contribute to the overall decrease in
weight gain in ob/ob-ChREBP⫺/⫺ mice, mice of the different
strains were fed a standard rodent chow diet without restriction
for 15 wk. Food consumption was measured daily, and the
mice were weighed every week. ob/ob-ChREBP⫺/⫺ mice
showed a dramatic reduction in weight gain compared with
ob/ob littermates at all time points (Fig. 2). Differences in
weight gain between the groups were directly related to food
intake. ob/ob mice consumed an average of 7.5 ⫾ 0.6 g of
chow daily, over one and one-half times the 4.4 ⫾ 0.1 and
4.7 ⫾ 0.1 g consumed daily by WT and ChREBP⫺/⫺ mice,
respectively. ob/ob-ChREBP⫺/⫺ mice averaged 5.8 ⫾ 0.2 g of
chow daily, which was 25% less than the ob/ob animals,
although still more than was eaten by either ChREBP⫺/⫺ or
WT mice. To investigate the effect of restricted food intake on
weight gain, 9-wk-old mice of all strains were fed 4.4 g/day of
standard chow, the amount naturally consumed by WT mice,
for 20 days, and their weights were measured daily. Only ob/ob
mice lost significant weight, ⬃20% of their initial weight
(Table 5), by caloric restriction. Weight loss in the ob/ob mice
occurred almost entirely in the first week of restricted food
intake and then stabilized, whereas the other groups maintained
their initial weights throughout (data not shown). Liver
triglyceride content was strikingly reduced in ob/ob mice
after 20 days of restricted food intake and modestly reduced
in WT mice, whereas there was essentially no change in the
liver triglyceride content of either ChREBP⫺/⫺ or ob/obChREBP⫺/⫺ mice.
Appetite-controlling hormones. Leptin modulates food consumption largely through its effects on appetite-controlling
neuropeptides (22). To investigate whether reduced food intake
in ob/ob-ChREBP⫺/⫺ mice might result from altered expression of these neuropeptides by leptin-independent mechanisms,
hypothalamic mRNA levels encoding appetite-stimulating or
orexic neuropeptides, NPY and AgRP, and the anorexic or
appetite-inhibiting peptides CART and POMC-derived ␣-melanocyte-stimulating hormone were determined (Fig. 3). In
ChREBP⫺/⫺ mice, mRNA levels of all of the neuropeptides
were not significantly different than those in WT mice. In
OB/OB
CHREBP DEFICIENCY PREVENTS OBESITY IN
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ChREBP activity is required for suppression of IRS-2 but not
PEPCK mRNA levels in chronically hyperinsulinemic ob/ob
mice. IRS-2 and IRS-1 mRNA levels in mice genetically
deficient only in ChREBP were not different from those in WT
mice. Taken together, these results suggest that downregulation
of IRS-2 expression in ob/ob mice occurs as a consequence of
hepatic metabolic derangements that are prevented by
ChREBP deficiency.
Increased mRNA expression of liver G-6-Pase but not
PEPCK was observed previously in ob/ob mice (26),
whereas elevated mRNA expression of both PEPCK and
G-6-Pase was found in an earlier study (30). Differences in
study conditions that may account for these divergent results
are not apparent, but it is noteworthy that liver PGC-1␣
mRNA levels in ob/ob mice in the latter study were also
elevated compared with control mice. In contrast, in ob/ob
mice described in this report, liver PGC-1␣ mRNA levels
were modestly decreased, and PEPCK but not G-6-Pase
mRNA expression paralleled PGC-1␣ mRNA levels in all
mice examined. Elevated G-6-Pase expression in ob/ob mice
is thus not dependent on elevated PGC-1␣ expression, as
was suggested previously (30).
Despite significantly lower plasma glucose levels in ob/obChREBP⫺/⫺ mice compared with ob/ob mice, there was no
corresponding decrease in plasma insulin levels. This observation is consistent with previous suggestions that hyperglycemia
is not the predominant cause of hyperinsulinemia in ob/ob mice
(1, 17). Severe peripheral insulin resistance in ob/ob and
ob/ob-ChREBP⫺/⫺ mice is likely to result, in large part, from
persistent hyperinsulinemia. It is perhaps not surprising, then,
that the apparent restoration of insulin-sensitive suppression of
liver gluconeogenic enzyme activity in ob/ob-ChREBP⫺/⫺
mice is not sufficient to fully normalize plasma glucose levels.
An additional role of ChREBP uncovered in this study was
that of appetite control. The elevated food intake of ob/ob mice
was significantly reduced by ChREBP deficiency and was
accompanied by reduced expression of the appetite-stimulating
neuropeptide AgRP and reversed by AgRP administration.
Expression of another appetite-stimulating hormone, NPY, was
somewhat increased in ob/ob-ChREBP⫺/⫺ compared with
ob/ob mice. However, although AgRP administration increases
cumulative food intake in mice, NPY administration acutely
increases food intake without altering cumulative consumption
(24). Thus the decrease in AgRP expression with ChREBP
deficiency in ob/ob mice is a likely mechanism contributing to
decreased food consumption and overall reduction in weight
gain in ob/ob-ChREBP⫺/⫺ mice. Whether ChREBP decficiency decreases AgRP expression in ob/ob mice indirectly or
ChREBP directly contributes to increased AgRP gene transcription in ob/ob mice will be the subject of future investigation.
In summary, ChREBP deficiency overcomes obesity in
ob/ob mice, reduces specific risk factors for obesity-associated
diseases, and improves appetite control, suggesting that reduction of ChREBP activity may have beneficial effects in the
treatment of obesity. The current findings suggest that the
physiological roles of ChREBP are broad and not limited to
liver. Continued investigation into ChREBP function in other
tissues as well as liver may uncover new and interesting
mechanisms in response to hormones and dietary nutrients.
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lipogenic enzymes and several key glycolytic enzymes essential for coordinated control of glucose metabolism and the
synthesis of fatty acids and triglycerides in vivo. The results
presented herein from ChREBP-deficient ob/ob mice extend
the important roles of ChREBP to the regulation of carbohydrate utilization and fat storage under conditions of caloric
excess.
In liver, ob/ob-ChREBP⫺/⫺ mice show phenotypic effects
quite similar to those in ChREBP⫺/⫺ mice (13) Decreased
induction of LPK and lipogenic enzyme gene expression resulted in markedly decreased hepatic fatty acid synthesis. The
near normalization of liver triglyceride content and plasma free
fatty acid and triglyceride levels in ob/ob-ChREBP⫺/⫺ mice
suggests that de novo synthesis of fatty acids from carbohydrate, rather than an increase in total intake of dietary fat, is the
predominant cause of elevated plasma triglycerides and fatty
liver development in ob/ob mice. Caloric restriction in ob/obChREBP⫺/⫺ mice did not result in any additional decrease in
hepatic liver triglycerides, in marked contrast to the greater
than 50% decrease in liver triglyceride content in ob/ob mice,
providing further evidence of the primary role of ChREBPregulated fatty acid synthesis in hepatic fat deposition. Epididymal and brown fat weight also were decreased in ob/obChREBP⫺/⫺ mice. It is not clear whether these effects are
secondary to decreased hepatic fatty acid synthesis or result
from a direct role of ChREBP in regulating fat synthesis in
these tissues. ChREBP expression is detectable in a number of
different tissues, albeit at much lower levels than in liver (29).
As observed previously, ChREBP deficiency leads to increased storage of liver glycogen (13). Decreased LPK expression in ChREBP-deficient mice results in lower glycolytic
activity and consequently higher liver glucose 6-phosphate
content; this ultimately leads to increased glycogen synthesis.
The much greater increase in liver glycogen content in ob/obChREBP⫺/⫺ mice compared with mice deficient solely in
ChREBP is likely to result both from greater reduction in
G-6-Pase expression, which limits glycogenolysis, and from
greater food intake in the doubly deficient mice.
Hyperglycemia in type 2 diabetes and other insulin resistance syndromes results in part from inappropriately high rates
of hepatic glucose synthesis and release. The loss of insulinsensitive repression of key gluconeogenic enzyme gene transcription as the result of downregulated IRS-2 expression is
one mechanism suggested to result in this selective hepatic
insulin resistance (21, 23, 27). In ob/ob mice in the studies
reported here, liver IRS-2 mRNA levels were lower and
G-6-Pase but not PEPCK mRNA levels were higher than those
in WT mice. ChREBP deficiency in ob/ob mice was associated
with normalization of liver IRS-2 and IRS-1 mRNA levels,
suppression of liver G-6-Pase mRNA to levels lower than those
in WT or ChREBP⫺/⫺ mice, and a further lowering of PEPCK
mRNA levels compared with modestly decreased levels in
ob/ob mice. Notably, the apparent effect of ChREBP deficiency in ob/ob mice to improve insulin-sensitive regulation of
liver gluconeogenesis occurred despite markedly elevated
plasma insulin levels that were not different from those in
ob/ob mice. Insulin represses IRS-2 gene transcription through
an insulin response element identical to that of the PEPCK
gene, and this mechanism was suggested as a potential cause of
decreased liver IRS-2 mRNA levels in chronic hyperinsulinemia (31). Results from the studies reported here indicate that
OB/OB
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CHREBP DEFICIENCY PREVENTS OBESITY IN
APPENDIX
14.
Table A1. Fasting glucose, insulin, and HOMA-IR
Glucose, mg/dl
Insulin, pmol/l
HOMA-IR
WT
ChREBP⫺/⫺
ob/ob
ob/ob-ChREBP⫺/⫺
15.
94.6⫾9.5
43.2⫾4.7
0.27⫾0.05
99⫾4.0
34.8⫾3.5
0.25⫾0.06
386⫾64
530⫾132
18.3⫾5.2
147⫾6.8
348⫾22
3.6⫾0.7
16.
Data are presented as means ⫾ SD. Homeostasis model assessment of
insulin resistance (HOMA-IR), a liver insulin sensitivity index, was calculated
as HOMA-IR ⫽ glucose (mg/dl)䡠insulin (␮U/ml)/405. Value more than 5
indicates the state of insulin resistance.
17.
18.
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22.
23.
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25.
26.
27.
28.
29.
30.
31.
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