Alternative Gnas gene products have opposite effects
on glucose and lipid metabolism
Min Chen*, Oksana Gavrilova†, Jie Liu*, Tao Xie*, Chuxia Deng‡, Annie T. Nguyen*, Lisa M. Nackers*, Javier Lorenzo*§,
Laura Shen*¶, and Lee S. Weinstein*?
*Metabolic Diseases Branch,†Mouse Metabolism Core Laboratory, and‡Genetics of Development and Disease Branch, National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
Edited by Lutz Birnbaumer, National Institutes of Health, Research Triangle Park, NC, and approved April 8, 2005 (received for review November 5, 2004)
Gnas is an imprinted gene with multiple gene products resulting
from alternative splicing of different first exons onto a common
exon 2. These products include stimulatory G protein ?-subunit
(Gs?), the G protein required for receptor-stimulated cAMP pro-
duction; extralarge Gs? (XL?s), a paternally expressed Gs? isoform;
and neuroendocrine-specific protein (NESP55), a maternally ex-
pressed chromogranin-like protein. Gs? undergoes tissue-specific
imprinting, being expressed primarily from the maternal allele in
certain tissues. Heterozygous mutation of exon 2 on the maternal
(E2m?/?) or paternal (E2?/p?) allele results in opposite effects on
energy metabolism. E2m?/?mice are obese and hypometabolic,
whereas E2?/p?mice are lean and hypermetabolic. We now stud-
ied the effects of Gs? deficiency without disrupting other Gnas
gene products by deleting Gs? exon 1 (E1). E1?/p?mice lacked the
E2?/p?phenotype and developed obesity and insulin resistance.
The lean, hypermetabolic, and insulin-sensitive E2?/p?phenotype
appears to result from XL?s deficiency, whereas loss of paternal-
specific Gs? expression in E1?/p?mice leads to an opposite meta-
bolic phenotype. Thus, alternative Gnas gene products have op-
posing effects on glucose and lipid metabolism. Like E2m?/?mice,
E1m?/?mice had s.c. edema at birth, presumably due to loss of
maternal Gs? expression. However, E1m?/?mice differed from
E2m?/?mice in other respects, raising the possibility for the
presence of other maternal-specific gene products. E1m?/?mice
had more severe obesity and insulin resistance and lower meta-
bolic rate relative to E1?/p?mice. Differences between E1m?/?and
E1?/p?mice presumably result from differential effects on Gs?
expression in tissues where Gs? is normally imprinted.
G protein ? genomic imprinting ? pseudohypoparathyroidism
for the intracellular cAMP response to hormones and other
extracellular signals (1). Heterozygous Gs? inactivating muta-
tions lead to Albright hereditary osteodystrophy (AHO), a
syndrome characterized by obesity and skeletal and neurobe-
havioral defects (1). Patients who inherit AHO maternally also
develop resistance to parathyroid hormone (PTH), thyroid-
stimulating hormone (TSH), and gonadotropins, a condition
known as pseudohypoparathyroidism type 1A. In contrast, pa-
tients who inherit AHO paternally do not develop multihormone
resistance (referred to as pseudopseudohypoparathyroidism)
manner, being primarily expressed from the maternal allele in
certain hormone-responsive tissues (2–5).
Gs? is encoded by a complex imprinted gene (GNAS at 20q13
in human, Gnas on mouse chromosome 2) that produces mul-
tiple gene products via the use of multiple alternative promoters
and first exons that splice onto a common set of downstream
exons (Fig. 1A) (1). Promoters for 55-kDa neuroendocrine-
specific protein (NESP55), a chromogranin-like protein, and
extralarge Gs? (XL?s), a neuroendocrine-specific Gs? isoform
with a long N-terminal extension encoded by its alternative first
exon, are located 47 and 35 kb upstream of the Gs? promoter,
he ubiquitously expressed stimulatory G protein ?-subunit
(Gs?) couples receptors to adenylyl cyclase and is required
respectively (6). NESP55 is unrelated to Gs?, and its entire
coding region is within its first exon; Gs? exons 2–13 are within
the 3? untranslated region of NESP55 transcripts (7). XL?s is
capable of mediating receptor-stimulated cAMP production in
transfected cells (8), and XL?s knockout mice (which also lack
a neural-specific isoform XLN1 and an alternative translation
product named ALEX) have a severe lean and insulin-sensitive
phenotype with early lethality (9). NESP55 and XL?s are
oppositely imprinted: NESP55 is expressed from the maternal
allele, and its promoter is DNA-methylated on the paternal
allele, whereas XL?s is paternally expressed, and its promoter
region is methylated on the maternal allele (6, 10). Exon 1A,
another alternative first exon located just upstream of the Gs?
promoter, is methylated on the maternal allele and generates
paternal-specific untranslated transcripts (11, 12). Loss of exon
1A imprinting leads to pseudohypoparathyroidism type 1B, a
syndrome of isolated parathyroid hormone resistance (12).
We previously generated mice with mutation of Gnas exon 2
(E2), an exon common to all Gnas transcripts, which can
potentially disrupt multiple Gnas gene products. Homozygotes
were embryonically lethal, whereas heterozygotes with mutation
on the maternal (E2m?/?) and paternal (E2?/p?) allele had
distinct phenotypes (referred to as m??? and ??p? in ref. 2).
E2m?/?mice had s.c. edema at birth, and most died between 1
and 3 weeks of age, after developing neurological signs. In
contrast, E2?/p?mice were small at birth, and most failed to
in mice with paternal and maternal uniparental disomy of distal
chromosome 2 where Gnas resides, suggesting that the pheno-
types result from loss of maternal- and paternal-specific Gnas
gene products, respectively (13, 14). Similar phenotypes were
also observed in mice with a heterozygous missense mutation in
exon 6 (15, 16). Surviving E2m?/?mice became obese, hypo-
metabolic, and hypoactive, whereas E2?/p?survivors had re-
duced adipocity, with increased metabolic rate, activity levels,
The opposite metabolic phenotypes in E2m?/?and E2?/p?mice
were associated with decreased and increased urinary norepi-
nephrine excretion, respectively, suggesting they might be due to
opposite changes in sympathetic activity (17).
which should specifically disrupt expression of Gs? but not
alternative Gnas gene products. Homozygotes were embryoni-
cally lethal. In contrast to XL?s knockout and E2?/p?mice,
heterozygotes with paternal E1 mutation (E1?/p?) had normal
survival and developed obesity and insulin resistance. The
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: Gs?, stimulatory G protein ?-subunit; XL?s, extralarge Gs? isoform; NESP55,
55-kDa neuroendocrine-specific protein; AHO, Albright hereditary osteodystrophy; BAT,
brown adipose tissue; WAT, white adipose tissue; TSH, thyroid-stimulating hormone; E1,
Gs? exon 1; E2, Gs? exon 2.
§Present address: Stanford University School of Medicine, Palo Alto, CA 94305.
¶Present address: Princeton University, Princeton, NJ 08544.
?To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
May 17, 2005 ?
vol. 102 ?
E2?/p?phenotype appears to result from XL?s deficiency.
Alternative Gnas gene products have opposing effects on glu-
cose and lipid metabolism. The E1m?/?and E2m?/?phenotypes
differed in several respects, raising the possibility of other
from E1?/p?mice, presumably because of differences in Gs?
expression in tissues where Gs? is normally imprinted.
Materials and Methods
Construction of Targeting Vector. A genomic clone including the
Gnas upstream region was isolated by screening a 129?SvEv
genomic library with a 1.4-kb probe containing E1. A 10.8-kb NotI
Complementary DNA linkers containing a loxP site (forward
TATACGAAGTTATGAGCTCCATATCG-3?) were ligated into
an NruI site at position ?419 (all positions are relative to Gs?
translational start site). A 3.1-kb BamH1 fragment (?4,728?
?1,601) was ligated into a HpaI site located upstream of the
loxP-neomycin resistance (Neo)-loxP cassette of pLoxpneo (20),
and a 5.6-kb BamH1 fragment (?1,601??3,962) was ligated into a
BamH1 site located downstream of loxP-Neo-loxP.
Generation of Gs? Knockout Mice. TC-1 ES cells (21) were elec-
troporated with the linearized targeting vector and selected with
G418 and 1-(2?-deoxy-2?-fluoro-?-D-arabinofuranosyl)-5-
iodouracil. Doubly resistant clones were screened for the
E1neo?flallele by Southern analysis by using 5? and 3? probes,
which were located outside the targeting construct (Fig. 1 B and
C). These cells were injected into C57BL?6 blastocysts im-
planted into pseudopregnant foster mothers. Three chimeric
mice (?70–80% derived from ES cells, as judged by coat color)
were bred with Black Swiss mice, and two had germ-line
transmission. F1mice were bred with EIIa promoter-cre trans-
genic mice (22), and mice with recombination at the most
upstream and downstream loxP sites were selected, resulting in
mice with deletion of E1 (E1?allele, Fig. 1B, referred to as
??1601/?419in ref. 23). E1?mice were mated with wild-type CD-1
mice (Charles River Laboratories) for several generations to
place them in the same genetic background as previously studied
E2?mice (2, 17–19). Subsequent genotyping was performed by
PCR of mouse tail DNA by using the common upstream primer
5?-GAGAGCGAGAGGAAGACAGC-3? and downstream
primers 5?-TCGGGCCTCTGGCGGAGCTT-3? and 5?-
AGCCCTACTCTGTCGCAGTC-3?, which generated 330- and
250-bp bands from the wild-type (E1?) and E1?alleles, respec-
tively. Primers correspond to GenBank accession no. AF152375,
bases 1293–1312, 1619–1600, and 3507–3488, respectively. An-
imals were maintained on a 12:12-h light?dark cycle and stan-
dard pellet diet (NIH-07, 5% fat by weight). Except when noted,
all experiments were performed on 12- to 14-week-old male
mutant mice and wild-type littermates. Experiments were ap-
proved by the National Institute of Diabetes and Digestive and
Kidney Diseases Animal Care and Use Committee.
Northern Analysis. Total tissue RNA was isolated by using TRIzol
reagent (Invitrogen). For Northern analysis, membranes were
hybridized with a32P-labeled E1-specific cDNA probe, which
was generated by PCR by using primers 5?-GGCAACAGTAA-
GACCGAGGACCAG-3? and 5?-ACCCAGCAGCAGCAG-
GCG-3? (GenBank accession no. AF152375, bases 3011–3034
and 3139–3122, respectively). Gs? signals were quantified by
using a Fuji BAS1500 phosphorimager and normalized to 18S
RNA, which was quantified by ethidium bromide by using
ALPHAEASE FC software (Alpha Innotech, San Leandro, CA).
Body Composition, Food Intake, Metabolic Activity, and Activity
Measurements. Body composition was measured by using the
Minispec mq10 NMR analyzer (Bruker Optics, The Woodlands,
TX). Food intake, metabolic rates (oxygen consumption rate by
and Gs? (1) splicing to a common set of exons (exons 2–12, not shown). Differentially methylated regions (Meth) and transcriptionally active promoters (arrows)
are indicated. The dashed arrow indicates that transcription from the paternal Gs? promoter is suppressed in some tissues. Antisense transcripts from the XL?s
promoter are not shown. The diagram is not drawn to scale. (B) The upstream portion of wild-type Gnas including exons 1A, 1, 2, and 3 is shown at the top. The
positions of the 5? and 3? probes used for Southern analysis are shown above. The scale above is in kb, with position 0 being the Gs? translational start site. A
loxP site (triangle) was inserted within an NruI site (N) at ?419, and upstream and downstream BamH1 (B) fragments were cloned into pLoxpneo to generate
the targeting construct. The E1neo?fland E1?alleles are shown below. S, SacI; Bg, BglII; H, HpaI; Neo, neomycin resistance gene; TK, thymidine kinase cassette.
(C) Southern analysis of genomic DNA from ES cells (two right lanes) or mice (two left lanes) after SacI digestion and hybridization with the 5? probe. Genotypes
are indicated above each lane. (D) Gs? mRNA levels (expressed as percent of wild-type) in liver (Left) and BAT (Right) from E1?/p?and E1m?/?mice (n ? 4–5 per
Generation of E1?mice. (A) The maternal (Mat) and paternal (Pat) alleles of Gnas are depicted with alternative first exons for NESP55 (NESP), XL?s, 1A,
Chen et al. PNAS ?
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vol. 102 ?
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indirect calorimetry), and activity levels were determined as
Blood and Tissue Analysis. Blood was obtained by retroorbital
bleed. Random serum glucose and triglyceride levels were
measured with an autoanalyzer by the National Institutes of
Health Clinical Chemistry Laboratory. Serum insulin, leptin,
glucagon, and adiponectin, and free T4 were measured by
radioimmunoassays (Linco Research, St. Charles, MO), and free
fatty acids were measured by using a free fatty acid kit (Roche
Diagnostics). TSH was measured by using a rat TSH assay kit
(Amersham Pharmacia). Liver triglycerides were measured by
solvent extraction followed by a radiometric assay for glycerol
(24). For histology, tissues were fixed in 10% neutral formalin
and paraffin-embedded, and sections were stained with hema-
toxylin?eosin. Urine was collected as described (17), and nor-
epinephrine was measured by HPLC (25).
Glucose and Insulin Tolerance Tests. i.p. glucose and insulin toler-
ance tests were performed in overnight-fasted male mice after
injection of glucose (2 mg?g) or insulin (Humulin, 0.75 milli-
units?g). Blood glucose values were obtained from tail-vein
bleeds by using a Glucometer Elite (Bayer, Elkhart, IN).
Statistical Analysis. Data are expressed as mean ? SEM. Statis-
or unpaired Student t test or multifactor ANOVA, with differ-
ences considered significant at P ? 0.05.
Generation of Mice with a E1 Deletion (E1?). To study mice with
specific disruption of Gs?, we generated mice in deletion of E1
(Fig. 1B). Correct targeting and subsequent cre-mediated re-
combination were confirmed by Southern analysis (Fig. 1 B and
by Northern analysis of liver and brown adipose tissue (BAT)
RNA from E1?/p?and E1m?/?mice, respectively (Fig. 1D).
As with E2?mice (2), matings between E1?heterozygotes
failed to produce homozygous E1?/?offspring, confirming that
edema at birth, which resolved within 1–2 days, a feature also
present in E2m?/?mice (2). E1m?/?mice had a 3-week expected
survival rate of 51% (27 E1m?/?vs. 53 E1?/?), a higher survival
rate than that observed in E2m?/?mice (2). Moreover, most
E2m?/?mice die between 1 and 2 weeks after birth (2), whereas
the survival loss of E1m?/?mice occurred within 3 days after
In contrast to both E2?/p?(2) and XL?s knockout (9) mice,
E1?/p?mice had normal survival (103 E1?/p?vs. 113 E1?/?mice
at 3 weeks) and no obvious preweaning phenotype. Moreover,
the phenotype observed in adult E2?/p?(2) was absent in adult
E1?/p?mice (see below). The similarity between E2?/p?and
XL?s knockout phenotypes and the absence of this phenotype
in E1?/p?mice confirm that the E2?/p?phenotype is a direct
result of XL?s, rather than Gs?, deficiency.
E1?/p?and E2?/p?Mice Have Opposite Metabolic Phenotypes. We
next examined whether there were differences in the metabolic
phenotype of adult E1?/p?and E2?/p?mice. In contrast to
E2?/p?mice (17), E1?/p?mice had normal body weight (Fig. 2A)
and increased fat mass based on NMR analysis and increased
BAT and white adipose tissue (WAT) pad weights (Fig. 2 B and
C). Interscapular fat pads of E1?/p?mice showed greater lipid
accumulation in both WAT and BAT (Fig. 2D). E1?/p?mice had
normal liver, heart, and spleen weights and a slight decrease in
kidney weights (Fig. 2B). Consistent with the fact that E1?/p?
mice have normal body weight, there were no differences in food
intake or resting and total metabolic rates between E1?/p?mice
and wild-type littermates (Fig. 3 A and B). There were also no
differences in total or ambulatory activity levels (Fig. 3D) or in
urinary norepinephrine excretion (E1?/p?389 ? 31 vs. wild-type
335 ? 31 nmol?nmol creatinine; n ? 5 per group). These results
are in contrast to those in E2?/p?mice, in which metabolic rate,
activity, and urinary norepinephrine were all increased (17).
E1?/p?mice had a small decrease in the respiratory exchange
quotient at 22°C (Fig. 3C).
Random-fed E1?/p?had normal serum glucose and elevated
serum insulin levels (Table 1). Glucose and insulin tolerance
tests showed E1?/p?mice to be both glucose-intolerant and
insulin-resistant (Fig. 4). This finding contrasts with E2?/p?
mice, which have increased glucose tolerance and insulin sensi-
tivity (18, 19). Random glucagon levels were unaffected in
E1?/p?mice (E1?/p?154 ? 46 vs. wild-type 99 ? 16 pg?ml; n ?
(mutants, filled bars; wild type, open bars) (n ? 6 per group). (B) Organ
weights in E1?/p?mice and wild-type littermates expressed as percent body
weight (n ? 6–9 per group). BAT, interscapular BAT; WAT, epidydimal WAT.
(C) Total, fat, and lean mass of E1?/p?(Left) and E1m?/?(Right) mice and their
(original magnification, ?100) of interscapular fat pads from E1?/p?(Left),
and WAT is below.*, P ? 0.05 vs. wild type.
Body and organ weights and body composition. (A) Body weights of
www.pnas.org?cgi?doi?10.1073?pnas.0408268102Chen et al.
5 per group). E1?/p?mice also showed changes in lipid metab-
olism that were opposite those found in E2?/p?mice. Serum
leptin levels were very low in E2?/p?mice (17) but were
significantly elevated in E1?/p?mice (Table 1). Because leptin
levels correlate with adiposity, increased leptin levels are con-
sistent with the increased fat mass of E1?/p?mice. E1?/p?mice
tended to have increased serum (Table 1) and liver triglyceride
levels (E1?/p?13.0 ? 2.2 vs. wild-type 9.2 ? 2.1 ?mol?g; n ? 8
per group) and had very reduced triglyceride clearance after an
intragastric lipid bolus (Fig. 5, which is published as supporting
information on the PNAS web site). Overall triglyceride metab-
olism in E1?/p?mice showed changes opposite those in E2?/p?
mice, which have reduced serum and liver triglyceride levels and
increased triglyceride clearance (17, 19). Serum free fatty acid
and adiponectin levels were unaffected in E1?/p?mice (Table 1).
E1m?/?Mice Have a More Severe Metabolic Phenotype than E1?/p?
Mice. Adult E1m?/?mice were 20% heavier than either E1?/p?
or wild-type mice (Fig. 2A), and virtually all of this increase was
accumulation in BAT and WAT (Fig. 2D). Consistent with more
severe obesity, E1m?/?mice had serum leptin levels that were
?3-fold higher than in wild-type mice and ?2-fold higher than
in E1?/p?mice (Table 1). Food intake was unaffected in E1m?/?
mice (Fig. 3A). However, unlike E1?/p?mice, E1m?/?mice had
a significantly lower resting metabolic rate at 23°C and 30°C and
total metabolic rate at 30°C (Fig. 3B). Activity levels also tended
to be lower in E1m?/?mice (Fig. 3D). Glucose and lipid
metabolism were also more severely affected in E1m?/?mice.
Serum glucose, insulin, and free fatty acid levels were signifi-
cantly higher in E1m?/?than in E1?/p?mice, and triglyceride
levels were higher in E1m?/?mice than in their wild-type
littermates (Table 1). E1m?/?mice were glucose-intolerant and
had much more severe insulin resistance than E1?/p?mice, with
virtually no hypoglycemic response to insulin (Fig. 4). Serum
adiponectin levels were unaffected in E1m?/?mice (Table 1).
Metabolic differences between E1m?/?and E1?/p?mice are not
due to hypothyroidism, because there are no differences in
group and wild-type mice (Table 1).
Because Gnas has multiple gene products, gene knockout mod-
els that specifically disrupt single gene products are required to
understand the biological role of each one. In this study, we
generated mice with specific loss of Gs? through deletion of its
specific first exon. Homozygotes are embryonically lethal, con-
firming that loss of Gs? cannot be compensated by other similar
genes, such as the olfactory G protein (26). Like E2m?/?mice (2)
and Oed mice with a maternal Gs? missense mutation, which
presumably disrupts Gs? function (15, 16), E1m?/?mice develop
s.c. edema at birth, confirming that this manifestation results
wild-type littermates (■) was measured at the indicated time points after
glucose (A) or insulin (B) administration (n ? 5–6 per group; results for insulin
are shown in Left and Right, respectively. The genotype had a significant
effect by two-factor ANOVA in all studies.
Glucose and insulin tolerance tests. Blood glucose in mutants (Œ) and
(Left) and E1m?/?(Right) mice (n ? 4–6 per group). (B) Resting and total
in E1?/p?(Upper) and E1m?/?(Lower) mice. (C) Respiratory exchange ratio
(RER) measured over 24 h at the indicated temperatures in E1?/p?(Left) and
per (group). Mutants and their respective wild-type littermates are shown as
filled and open bars, respectively.*, P ? 0.05 vs. wild type.
Food intake, metabolic rate, and activity. (A) Food intake in E1?/p?
Chen et al. PNAS ?
May 17, 2005 ?
vol. 102 ?
no. 20 ?
from loss of maternal Gs? expression (see Table 2 for a summary
of Gnas knockout models). That edema is absent in E1?/p?mice
suggests that this manifestation results from severe Gs? defi-
ciency in one or more tissues where Gs? is normally paternally
imprinted. Several imprinted genes have been shown to affect
placental function. Additional studies are necessary to deter-
mine whether Gs? imprinting in placenta could account for the
s.c. edema during late gestation observed in several maternal
Gs? knockout models.
However, E1m?/?and E2m?/?phenotypes differ in many
obesity compared with E2m?/?mice and lack the neurological
defects often observed in 1- to 3-week-old E2m?/?mice (2).
Moreover, whereas E2m?/?mice have increased insulin sensi-
tivity (18), E1m?/?are severely insulin-resistant. The more
severe obesity of E1m?/?mice likely contributes to their insulin
resistance. However, Gs? deficiency is also an important factor,
because E1?/p?mice, which are only mildly obese, are also
insulin-resistant. Disruption of other maternal-specific Gnas
gene products in E2m?/?, but not E1m?/?, mice is the most likely
explanation for the phenotypic differences between these two
mutant lines. However, the only other known maternal-specific
Gnas gene product, NESP55, is not disrupted in E2m?/?mice
(T.X., data not shown), and NESP55 knockout mice do not have
altered viability or metabolism (27). Moreover, loss of NESP55
expression in some pseudohypoparathyroidism type IB patients
does not lead to an obvious phenotype (12). Our attempts to
identify other maternal Gnas products by 5? RACE have been
unsuccessful. Another partial maternal-specific mRNA tran-
script has been identified (28), although it is unknown whether
Although both E1m?/?and E2m?/?mice were studied in a CD-1
background, we cannot exclude the possibility that genetic
background differences contributed to phenotypic differences
observed between these two lines.
The absence of early lethality or lean phenotype in E1?/p?
mice confirms that these manifestations in E2?/p?mice are not
caused by Gs? deficiency. XL?s knockout mice show a pheno-
type very similar to E2?/p?mice (9), indicating that the effects
in the latter result from complete loss of paternally expressed
XL?s. Moreover, adult XL?s knockout mice have the same
(T.X., L.S.W., A. Plagge, and G. Kelsey, unpublished results).
Therefore, the E2?/p?phenotype at all stages of life is deter-
mined by XL?s deficiency, and XL?s deficiency appears to be
dominant over paternal-specific Gs? deficiency. The opposite
metabolic phenotypes of E2?/p?and XL?s knockout mice vs.
E1?/p?and E1m?/?mice demonstrate that Gs? and XL?s have
opposing effects on glucose and lipid metabolism. Although it
has been suggested that Gs? and XL?s may directly counteract
each other biochemically (9), this has not been established, and
studies examining the role of XL?s in receptor-mediated sig-
naling have provided conflicting results (8, 29). We have pro-
posed that XL?s may normally inhibit sympathetic activity in the
central nervous system, and that XL?s deficiency therefore leads
to increased sympathetic activity (19). Whatever the mechanism,
Table 1. Serum chemistries in E1?/p?and E1m?/?mice and wild-type littermates
Free fatty acids, nM
Free T4, ng?dl
180 ? 9
2.1 ? 0.3
0.410 ? 0.034
173 ? 19
10.6 ? 4.3
6.30 ? 0.58
0.65 ? 0.09
1.51 ? 0.04
165 ? 5
5.7 ? 1.9*
0.412 ? 0.032
215 ? 31
16.4 ? 2.2*
6.87 ? 0.53
0.62 ? 0.09
1.36 ? 0.03
156 ? 18
3.2 ? 1.6
0.468 ? 0.066
122 ? 12
14.4 ? 4.7
6.89 ? 1.57
0.71 ? 0.16
1.40 ? 0.09
209 ? 21†
44.9 ? 20.7†
0.675 ? 0.079*†
200 ? 25*
39.2 ? 6.0*†
6.97 ? 0.65
0.84 ? 0.06
1.42 ? 0.08
n ? 5–22 for E1?/p?mice and controls; n ? 5–8 for E1m?/?mice and controls. *, P ? 0.05 vs. ?/?. †, P ? 0.05
Table 2. Summary of Gnas knockout mouse lines
Mouse line (source)Genetic deficienciesPhenotypes
E2m???(2, 17, 18)Maternal Gs?
Possibly other maternal products
s.c. edema at birth
Severe preweaning lethality
Obesity; increased insulin sensitivity
E2??p?(2, 17–19)Paternal Gs?; XL?s Perinatal lethality
Very lean, greatly increased insulin sensitivity
Oed (maternal mutation) (15, 16) Maternal Gs?
Possibly other maternal products
s.c. edema at birth
Severe preweaning lethality
Increased BAT mass
Sml (paternal mutation) (15, 16)Paternal Gs?; XL?s Similar to E2??p?
XL?s knockout (9)XL?s, XLN1, ALEXSimilar to E2??p?
NESP55 knockout (27)NESP55Normal viability and metabolism
E1m???(this study)Maternal Gs?
s.c. edema at birth
Moderate preweaning lethality (50%)
Severe obesity, insulin resistance
E1??p?(this study) Paternal Gs?
Obesity, insulin resistance
www.pnas.org?cgi?doi?10.1073?pnas.0408268102Chen et al.
the role of XL?s in metabolic regulation appears to be species-
specific, because pseudopseudohypoparathyroidism patients
with paternal GNAS mutations that disrupt XL?s expression do
not develop a similar phenotype (1).
The presence of obesity in both E1m?/?and E1?/p?mice
provides further evidence that the obesity in AHO patients
results from Gs? deficiency. This hypothesis is further supported
by the fact that patients with E1 mutations, which disrupt only
Gs? expression, have the same phenotype as patients who inherit
mutations in downstream exons common to all GNAS tran-
scripts. Because Gs? is ubiquitously expressed and mediates
important signals for metabolic regulation in almost all tissues,
it is unclear whether the obesity in heterozygous E1?mice and
AHO patients results from one specific metabolic defect or is the
cumulative result of subtle metabolic defects in multiple tissues.
It is unlikely that obesity in E1?heterozygotes is solely due to the
resistance of fat cells to sympathetic- or hormone-mediated
metabolic activation, because E2m?/?mice have a normal met-
abolic response to a ?3 adrenergic agonist, which primarily
targets adipose tissue (17), and preliminary observations show
that mice heterozygous for an adipose tissue-specific Gs? knock-
out do not develop significant obesity (M.C. and L.S.W., un-
to their insulin resistance, although direct effects of partial Gs?
deficiency on insulin sensitivity cannot be ruled out.
In many respects (s.c. edema at birth, early lethality, more
severe obesity, and insulin resistance), E1m?/?mice have a more
severe phenotype than E1?/p?mice. These differences are
presumably the consequence of differential expression of Gs? in
tissues where Gs? is normally preferentially expressed from the
maternal allele, leading to tissue-specific Gs? deficiency in
E1m?/?, but not E1?/p?, mice. This hypothesis is supported by
the observation that many features of the E1m?/?phenotype are
reversed by paternal deletion of the exon 1A region, a region
required for tissue-specific paternal Gs? imprinting (30, 31).
Although it is possible that maternal effects during gestation
could contribute to the E1m?/?phenotype, the phenotype of
E1m?/?offspring is not affected by whether the mutant mother
is E1m?/?or E1?/p?.
The greater severity of the metabolic phenotype in E1m?/?as
compared with E1?/p?mice likely results from more severe Gs?
deficiency in one or more tissues involved in metabolic regula-
tion. Given that metabolic rates are reduced in E1m?/?, but not
E1?/p?, mice, one hypothetical mechanism is reduced hypotha-
lamic melanocortin-Gs? signaling in E1m?/?mice due to the
absence of Gs? resulting from maternal mutation and paternal
imprinting. However, it is presently unknown whether Gs? is
imprinted within the hypothalamus. Although pseudohypopara-
thyroidism type 1A patients develop TSH resistance, thyroid
function is normal in E1m?/?mice, and therefore hypothyroid-
ism is not a cause of their reduced metabolic rates. Additional
studies in this and tissue-specific Gs? knockout models will
provide a greater understanding of the role of the Gnas locus in
development and metabolic regulation.
We thank I. P. Blazicek, K. Pacak, S. Pack, and R. Vinitsky for technical
1. Weinstein, L. S., Yu, S., Warner, D. R. & Liu, J. (2001) Endocr. Rev. 22, 675–705.
2. Yu, S., Yu, D., Lee, E., Eckhaus, M., Lee, R., Corria, Z., Accili, D., Westphal,
H. & Weinstein, L. S. (1998) Proc. Natl. Acad. Sci. USA 95, 8715–8720.
3. Liu, J., Erlichman, B. & Weinstein, L. S. (2003) J. Clin. Endocrinol. Metab. 88,
4. Hayward, B. E., Barlier, A., Korbonits, M., Grossman, A. B., Jacquet, P.,
Enjalbert, A. & Bonthron, D. T. (2001) J. Clin. Invest. 107, R31–R36.
5. Mantovani, G., Ballare, E., Giammona, E., Beck-Peccoz, P. & Spada, A. (2002)
J. Clin. Endocrinol. Metab. 87, 4736–4740.
6. Hayward, B. E., Moran, V., Strain, L. & Bonthron, D. T. (1998) Proc. Natl.
Acad. Sci. USA 95, 15475–15480.
7. Weiss, U., Ischia, R., Eder, S., Lovisetti-Scamihorn, P., Bauer, R. & Fischer-
Colbrie, R. (2000) Neuroendocrinology 71, 177–186.
8. Bastepe, M., Gunes, Y., Perez-Villamil, B., Hunzelman, J., Weinstein, L. S. &
Ju ¨ppner, H. (2002) Mol. Endocrinol. 16, 1912–1919.
9. Plagge, A., Gordon, E., Dean, W., Boiani, R., Cinti, S., Peters, J. & Kelsey, G.
(2004) Nat. Genet. 36, 818–826.
10. Peters, J., Wroe, S. F., Wells, C. A., Miller, H. J., Bodle, D., Beechey, C. V.,
Williamson, C. M. & Kelsey, G. (1999) Proc. Natl. Acad. Sci. USA 96,
11. Liu, J., Yu, S., Litman, D., Chen, W. & Weinstein, L. S. (2000) Mol. Cell. Biol.
12. Liu, J., Litman, D., Rosenberg, M. J., Yu, S., Biesecker, L. G. & Weinstein, L. S.
(2000) J. Clin. Invest. 106, 1167–1174.
13. Cattanach, B. M. & Kirk, M. (1985) Nature 315, 496–498.
14. Williamson, C. M., Beechey, C. V., Papworth, D., Wroe, S. F., Wells, C. A.,
Cobb, L. & Peters, J. (1998) Genet. Res. 72, 255–265.
15. Cattanach, B. M., Peters, J., Ball, S. & Rasberry, C. (2000) Hum. Mol. Genet.
16. Skinner, J. A., Cattanach, B. M. & Peters, J. (2002) Genomics 80, 373–375.
17. Yu, S., Gavrilova, O., Chen, H., Lee, R., Liu, J., Pacak, K., Parlow, A. F., Quon,
M. J., Reitman, M. L. & Weinstein, L. S. (2000) J. Clin. Invest. 105, 615–623.
18. Yu, S., Castle, A., Chen, M., Lee, R., Takeda, K. & Weinstein, L. S. (2001)
J. Biol. Chem. 276, 19994–19998.
19. Chen, M., Haluzik, M., Wolf, N. J., Lorenzo, J., Dietz, K. R., Reitman, M. L.
& Weinstein, L. S. (2004) Endocrinology 145, 4094–4102.
20. Yang, X., Li, C., Xu, X. & Deng, C. (1998) Proc. Natl. Acad. Sci. USA 95,
21. Deng, C., Wynshaw-Boris, A., Zhou, F., Kuo, A. & Leder, P. (1996) Cell 84,
22. Xu, X., Li, C., Garrett-Beal, L., Larson, D., Wynshaw-Boris, A. & Deng, C. X.
(2001) Genesis 30, 1–6.
23. Sakamoto, A., Liu, J., Greene, A., Chen, M. & Weinstein, L. S. (2004) Hum.
Mol. Genet. 15, 819–828.
24. Burant, C. F., Sreenan, S., Hirano, K., Tai, T. A., Lohmiller, J., Lukens, J.,
Davidson, N. O., Ross, S. & Graves, R. A. (1997) J. Clin. Invest. 100,
25. Eisenhofer, G., Goldstein, D. S., Stull, R., Keiser, H. R., Sunderland, T.,
Murphy, D. L. & Kopin, I. J. (1986) Clin. Chem. 32, 2030–2033.
26. Jones, D. T., Masters, S. B., Bourne, H. R. & Reed, R. R. (1990) J. Biol. Chem.
27. Plagge, A., Isles, A. R., Gordon, E., Humby, T., Dean, W., Gritsch, S.,
Fischer-Colbrie, R., Wilkinson, L. S. & Kelsey, G. (2005) Mol. Cell. Biol. 25,
28. Holmes, R., Williamson, C., Peters, J., Denny, P., Wells, C., RIKEN GER &
GSL Members (2003) Genome Res. 13, 1410–1415.
29. Klemke, M., Pasolli, H. A., Kehlenbach, R. H., Offermanns, S., Schultz, G. &
Huttner, W. B. (2000) J. Biol. Chem. 275, 33633–33640.
30. Liu, J., Chen, M., Deng, C., Bourc’his, D., Nealon, J. G., Erlichman, B., Bestor,
T. H. & Weinstein, L. S. (2005) Proc. Natl. Acad. Sci. USA 102, 5513–5518.
31. Williamson, C. M., Ball, S. T., Nottingham, W. T., Skinner, J. A., Plagge, A.,
Turner, M. D., Powles, N., Hough, T., Papworth, D., Fraser, W. D., et al. (2004)
Nat. Genet. 36, 894–899.
Chen et al.PNAS ?
May 17, 2005 ?
vol. 102 ?
no. 20 ?