From the Metabolic Diseases Branch and
§ Diabetes Branch, NIDDK, National Institutes of Health,
Bethesda, Maryland 20892
Received for publication, November 13, 2000, and in revised form, March 13, 2001
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ABSTRACT |
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The stimulatory guanine nucleotide-binding
protein (Gs) is required for
hormone-stimulated cAMP generation. Gnas, the gene encoding
the Gs Heterotrimeric guanine nucleotide-binding proteins (G
proteins)1 transduce signals
from seven transmembrane receptors to intracellular effectors. Each G
protein is composed of distinct Heterozygous inactivating mutations of the gene encoding
Gs GNAS1 (and the murine homolog Gnas) are now known
to produce two additional gene products by use of alternative promoters and first exons, which splice onto a common second exon. XL We generated mice with an insertional disruption of Gnas
exon 2 (GsKO), an exon common to all known Gnas transcripts
(17). Homozygotes ( Mice--
Mice with insertion of a neomycin resistance cassette
into exon 2 of Gnas were previously created by targeted
mutagenesis (17). Female mutants were mated to wild type CD-1 males
(Charles River) to generate m Glucose and Insulin Tolerance Tests--
For the measurement of
fasting serum glucose and insulin levels, animals were
sacrificed after an overnight fast (16 h), and serum was collected.
Serum insulin was measured by radioimmunoassay (Linco Research
Immunoassay, St. Charles, MO). Serum glucose was measured using
the glucose HK kit (Sigma). For glucose tolerance tests, mice were
fasted overnight and anesthetized with an intraperitoneal injection of
pentobarbital (35 µg/g of body weight) and ketamine (70 µg/g of
body weight). Glucose (2.5 mg/g of body weight in 0.9% NaCl solution)
was administered intraperitoneally, and blood was collected from the
retroorbital veins in heparinized capillary tubes at time 0 (prior to
injection) and at 20, 40, 60, and 120 min after injection. Glucose was
measured using the glucose HK kit (Sigma). For insulin tolerance tests,
fasted mice were anesthetized with either pentobarbital and ketamine or
pentobarbital alone. Human regular insulin (Humulin, 0.75 mIU/g of body
weight in 0.9% NaCl solution) was administered intraperitoneally,
blood was collected at 0, 15, 30, 60, 90, and 120 min, and glucose was
measured using either the glucose HK kit or the Lifescan Surestep
glucometer (Johnson & Johnson). Anesthesia had no significant effect on
base-line glucose levels or response to insulin.
Measurement of 2-Deoxyglucose Uptake in Isolated Skeletal
Muscle--
In vitro glucose uptake into isolated extensor
digitorum longus muscle was measured using
[3H]2-deoxyglucose, with [14C]mannitol to
correct for extracellular fluid (30). Briefly, muscles were isolated,
allowed to recover for 3 h in Krebs-Heinseleit buffer containing
glucose (6 mM), mannitol (18 mM), and pyruvate (2 mM) in a 95% O2, 5% CO2
atmosphere, and then incubated in the presence or absence of 10 milliunits/ml insulin (Humulin, Eli Lilly) for 20 min, followed by
addition of 100 µM (2.4 µCi)
[1,2-3H]2-deoxyglucose and 0.7 µCi of
[1-14C]mannitol. After 20 min at 30 °C, the tissue
3H and 14C were measured, and the tissue
3H uptake into fibers was determined after adjustment for
14C uptake into extracellular spaces.
Measurement of Total GLUT4 Expression in Skeletal
Muscle--
Anterior tibialis muscles were dissected and immediately
frozen in liquid nitrogen. Muscles were weighed, homogenized on ice with a Teflon pestle at 1:10 (w/v) in HEPES-EDTA-sucrose buffer (20 mM HEPES, 1 mM EDTA, 250 mM
sucrose, pH 7.4, containing protease inhibitors (Complete capsules,
Roche Molecular Biochemicals)), and solubilized in 2.3 M
urea, 1.43% sodium dodecyl sulfate, 14.3 mM Tris, pH 6.8, and 100 mM dithiothreitol (all final concentrations). Samples were subjected to 10% SDS-polyacrylamide gel electrophoresis (Novex). After confirming equal loading of lanes by staining the gel
with Ponceau S, proteins were transferred to nitrocellulose membranes
by electroblotting. Filters were blocked, incubated with rabbit
polyclonal antibody raised to the carboxyl terminus of GLUT4
(1:500 dilution), and then incubated with 0.25 µCi/ml [125I]protein A (PerkinElmer Life Sciences) as
previously described (31). Bands were quantified using a
phosphorimaging device (Fuji BAS1000).
Statistical Analysis--
All values are reported as the mean ± S.E. Statistical significance was determined using the paired
t test or multi-factor ANOVA, with differences considered
significant at p < 0.05 (2-tailed).
To determine whether insulin sensitivity is altered in adult +/p-subunit, is imprinted, and targeted disruption of this gene in mice leads to distinct phenotypes in heterozygotes depending on whether the maternal (m
/+) or paternal (+/p
) allele is
mutated. Notably, m
/+ mice become obese, whereas +/p
mice are
thinner than normal. In this study we show that despite these opposite
changes in energy metabolism, both m
/+ and +/p
mice have greater
sensitivity to insulin, with low to normal fasting glucose levels, low
fasting insulin levels, improved glucose tolerance, and exaggerated
hypoglycemic response to administered insulin. The combination of
increased insulin sensitivity with obesity in m
/+ mice is unusual,
because obesity is typically associated with insulin resistance. In
skeletal muscles isolated from both m
/+ and +/p
mice, the basal
rate of 2-deoxyglucose uptake was normal, whereas the rate of
2-deoxyglucose uptake in response to maximal insulin stimulation was
significantly increased. The similar changes in muscle sensitivity to
insulin in m
/+ and +/p
mice may reflect the fact that muscle
Gs
expression is reduced by ~50% in both groups of
mice. GLUT4 expression is unaffected in muscles from +/p
mice.
Increased responsiveness to insulin is therefore the result of altered
insulin signaling and/or GLUT4 translocation. This is the first direct
demonstration in a genetically altered in vivo model that
Gs-coupled pathways negatively regulate insulin signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
,
, and
subunits (1). The
-subunit binds guanine nucleotide and interacts with specific
effectors, such as adenylyl cyclase, phospholipase C, and ion channels.
The
-subunit for Gs (Gs
) is ubiquitously expressed and transmits the stimulatory signal from hormone-bound receptors to adenylyl cyclase and is therefore required for
hormone-stimulated cAMP generation. Both catecholamines (whose
receptors activate Gs) and cAMP have been implicated as
negative regulators of insulin signaling and insulin-stimulated glucose
transport in various cell types, including adipocytes and muscle cells
(2-6), although this has not been found universally (7-9). In one
study administration of cholera toxin (which constitutively activates
Gs
) to rats led to decreased insulin sensitivity and
glucose uptake in skeletal muscle (10).
(GNAS1 at 20q13.2-13.3; Ref. 11) cause
Albright hereditary osteodystrophy, an autosomal dominant
disorder characterized by obesity, short stature, and skeletal defects
(12). Paternal transmission of GNAS1 mutations produces
offspring with Albright hereditary osteodystrophy alone
(pseudopseudohypoparathyroidism), whereas maternal transmission
produces offspring who also have multihormone resistance (termed
pseudohypoparathyroidism type Ia) (13), suggesting that the
Gs
gene is imprinted. Genomic imprinting is an
epigenetic phenomenon characterized by parental allele-specific
differences in gene expression (14-16). In mice Gs
is
expressed primarily from the maternal allele in some tissues, such as
renal proximal tubules and adipose tissue, but is biallelically expressed in most other tissues (17, 18). Although this could explain
the presence of hormone resistance in pseudohypoparathyroidism type Ia
and its absence in pseudopseudohypoparathyroidism, the imprinting of
Gs
in humans has yet to be directly demonstrated (19).
s, a
novel isoform of Gs
, is expressed exclusively from the
paternal allele, whereas NESP55, a chromogranin-like neurosecretory
protein, is expressed exclusively from the maternal allele (20-22).
Both are expressed primarily in neuroendocrine tissues, and their
biological function has not been elucidated (23-27). XL
s has been
shown recently to be able to activate adenylyl cyclase in
vitro but does not appear to be activated by
Gs-coupled receptors (28, 29).
/
) die during early postimplantation
development. Heterozygotes with disruption of the paternal (+/p
) or
maternal (m
/+) allele have distinct phenotypic manifestations (17), leading to early death in the majority of heterozygotes. m
/+ and
+/p
mice that survive past weaning have diametrically opposite abnormalities in fat mass, resting metabolic rate, and activity levels
(18). Adult +/p
mice are lean, hypermetabolic, and hyperactive, whereas m
/+ mice are obese and have lower resting metabolic rate and
activity levels. These effects may be due to increased and decreased
sympathetic activity in +/p
and m
/+ mice, respectively. In this
report we demonstrate that both lean +/p
mice and obese m
/+ mice
are more sensitive to insulin than normal mice, with increased
insulin-stimulated glucose uptake in skeletal muscle. This is the first
direct demonstration in a genetically altered in vivo model
that Gs-coupled pathways negatively regulate insulin signaling.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
/+ mice, and male mutants were mated to
wild type CD-1 females to generate +/p
mice. Wild type littermates of
m
/+ and +/p
mice are designated m+/+ and +/p+ mice, respectively. Animals were maintained on a 12-h light/dark cycle (6 a.m./6 p.m.) and
a standard pellet diet (NIH-07, 5% fat by weight).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and/or m
/+ mice, we measured glucose and insulin in fasted adult mice
and glucose after intraperitoneal administration of glucose or insulin.
Both +/p
and m
/+ mice had significantly lower fasting serum
insulin levels than their wild type littermates (Table
I). For +/p
mice five of six animals
had fasting insulin levels below the detection limit of the assay (0.05 ng/ml). Simultaneous glucose levels were less than or equal to those of
wild type littermates (Table I).
Fasting serum glucose and insulin
and m
/+ mice, respectively. Results are
expressed as the mean ± S.E. (n).
Glucose tolerance tests were next performed in 10-week-old male mice.
Glucose levels were lower in both +/p and m
/+ mice compared with
wild type littermates at all time points after injection of glucose
(2.5 mg/g of body weight), with the genotype having a significant
effect on the overall handling of glucose load (Fig. 1A). An overall decrease in
the glucose curve was observed in both groups of mutant mice, although
the return toward base-line glucose levels was more pronounced in +/p
mice. This difference in glucose tolerance between +/p
and m
/+ mice
might be predicted, given that +/p
mice are lean, whereas m
/+ mice
are obese. We next examined the glucose response to administered
insulin (0.75 mIU/g of body weight, intraperitoneal) (Fig.
1B). In this study base-line glucose was similar in all
groups. In both +/p
and m
/+ mice the hypoglycemic response to
injected insulin was significantly greater than in wild type
littermates.
|
Therefore, both +/p and m
/+ mice have increased insulin
sensitivity, based on the findings of lower fasting insulin levels with
normal or low fasting glucose, improved glucose tolerance, and a
greater than normal acute hypoglycemic response to insulin. Increased
insulin sensitivity might be expected in the lean +/p
mice, because
insulin sensitivity is generally inversely related to adiposity.
However, in m
/+ mice increased insulin sensitivity is associated with
obesity. The dissociation of obesity and insulin resistance has been
described previously in a few other animal models (e.g. mice
with a knockout of the adipocyte-specific aP2 gene; Ref. 32). The fact
that m
/+ are obese whereas +/p
are lean may explain why
glucose tolerance and insulin sensitivity appear to be somewhat greater
in +/p
mice as compared with m
/+ mice (Fig. 1).
Most of the acute hypoglycemic response to insulin is normally due to
increased glucose uptake in skeletal muscle, associated with
translocation of the insulin-responsive glucose transporter GLUT4 from
internal vesicles to the plasma membrane (33). Skeletal muscle
(extensor digitorum longus) was removed from +/p and m
/+ mice and
their wild type littermates, and the rates of 2-deoxyglucose uptake
were measured in the presence of either no insulin (Fig. 2, Basal) or a maximal
concentration of insulin (10 milliunits/ml; Fig. 2,
Insulin). In the absence of insulin, rates of 2-deoxyglucose uptake were similar in muscles from +/p
, m
/+, and wild type mice.
In contrast, in the presence of insulin the rates of 2-deoxyglucose uptake were significantly higher in muscles from both +/p
and m
/+
mice when compared with those from wild type mice. The maximal responses to insulin (increase over basal) were 55 and 48% greater in
muscles from +/p
and m
/+ mice, respectively, than the response in
muscles from wild type mice. These large differences in
insulin-stimulated glucose uptake in skeletal muscle largely account
for the greater hypoglycemic effect of insulin in +/p
and m
/+
mice.
|
One potential mechanism for the observed increase in insulin-stimulated
glucose uptake in skeletal muscle is increased expression of GLUT4 in
this tissue. cAMP has been shown to negatively regulate the GLUT4
promoter (34, 35), and therefore decreased expression of
Gs might be expected to lead to GLUT4 overexpression due
to decreased intracellular cAMP concentrations. However, immunoblotting of whole homogenates of skeletal muscle (anterior tibialis) with a
GLUT4-specific antibody showed that GLUT4 expression was unaffected in
skeletal muscles of +/p
mice (99 ± 15% of the level in
paired wild type littermates; n = 4 pairs; see Fig.
3).
|
Decreased levels of Gs expression and/or intracellular
cAMP in skeletal muscle of m
/+ and +/p
mice probably lead to
increased insulin-stimulated glucose uptake either by increasing the
responsiveness of the proximal insulin-signaling pathway to insulin or
by increasing GLUT4 translocation or transporter activity. Several
studies show that catecholamines or cAMP analogues can lead to
decreased insulin binding or decreased insulin receptor kinase activity
in response to insulin in cultured cells (3, 36-47), although some
studies failed to show an effect of these agents on insulin receptor
function (48-50). One potential mechanism for these effects is through
serine/threonine phosphorylation of the insulin receptor by
cAMP-dependent protein kinase (protein kinase A) (40, 46,
51). Serine/threonine phosphorylation of downstream components, such as
IRS-1 and -2, may decrease the ability of these molecules to be
activated by the insulin receptor (52). We are presently embarking on
studies to directly examine the ability of insulin to activate the
insulin receptor and other proximal insulin-signaling components in
tissues from Gnas knockout mice. There is also evidence that
catecholamines or cAMP may have a direct effect on GLUT4 transporter,
perhaps through decreasing the accessibility of GLUT 4 to the cell
surface (4, 53, 54). One recent study suggests that the rate of fusion
of GLUT4-containing vesicles to the plasma membrane in the presence of
insulin is reduced by catecholamines (4).
Although in many respects +/p and m
/+ have distinct phenotypes,
both groups of mice seem to have similar changes in insulin sensitivity, despite the fact that one group is leaner than normal and
the other is obese. We think this is most likely due to the fact that
Gs
is not imprinted in skeletal muscle, and therefore its expression in skeletal muscle is decreased to a similar extent in
+/p
and m
/+ mice (18). Gnas also produces two other
imprinted gene products, NESP55, a chromogranin-like protein that is
expressed only from the maternal allele, and XL
s, an alternative
isoform of Gs
with a long amino-terminal extension that
is only expressed from the paternal allele (20, 21). It would seem
unlikely that loss of these products leads to the observed increase in insulin sensitivity, because XL
s expression is lost only in +/p
mice, whereas NESP55 appears to be expressed in both m
/+ and +/p
mice.2 Moreover, we
could not detect expression of either of these gene products in
skeletal muscle of normal mice by reverse
transcription-polymerase chain reaction (data not shown).
Hormones that counterregulate insulin, including catecholamines and
glucagon, activate Gs-coupled pathways. Therefore
Gs deficiency might be expected to produce an apparent
increase in insulin sensitivity due to resistance to counterregulatory
hormones. However, in patients with pseudohypoparathyroidism type Ia
the glycemic response to maximal glucagon stimulation is normal (55, 56). In our experiments the time course of the response to administered insulin in +/p
and m
/+ mice is consistent with a direct effect on
skeletal muscle glucose utilization independent of the effects of
counterregulatory hormones. This was confirmed in our studies that
directly examined insulin-stimulated glucose uptake in skeletal muscle
in the absence of counterregulatory hormones.
Although our studies suggest that Gs may negatively
regulate insulin-stimulated glucose uptake, evidence suggests that two other heterotrimeric G proteins, namely Gq/11 and
Gi2, have the opposite effect on the metabolic response to
insulin. Two studies in 3T3-L1 adipocytes demonstrated that
Gq/11
is required for insulin-stimulated GLUT4
translocation (57, 58). One study suggested that Gq/11
might work upstream of phosphoinositide 3'-OH kinase and be
tyrosine phosphorylated by the insulin receptor (57), whereas the other
study suggested that Gq/11
works downstream of
phosphoinositide 3'-OH kinase (58). Mice in which
Gi2
expression was decreased in liver and adipose tissue
using an antisense RNA were insulin-resistant (59), whereas those
expressing activated Gi2
had increased insulin
sensitivity (60-62). Gi2
appears to have biological
effects opposite those of Gs
in several systems (63,
64). Further studies are required to determine whether Gs
inhibits insulin signaling directly or through the
actions of cAMP and/or cAMP-dependent protein kinase and
what steps in the insulin receptor-GLUT4 pathway are affected by
decreased Gs
expression.
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ACKNOWLEDGEMENTS |
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We thank Sam Cushman and Dena Yver for their technical advice and assistance and for providing the GLUT4 antibody.
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FOOTNOTES |
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* 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.
¶ To whom correspondence should be addressed: Bldg. 10, Rm. 8C101, National Institutes of Health, Bethesda, MD 20892-1752. Tel.: 301-402-2923; Fax: 301-402-0374; E-mail: leew@amb.niddk.nih.gov.
Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M010313200
2 S. Yu and L. S. Weinstein, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: G protein, guanine nucleotide-binding protein; Gs, stimulatory G protein; ANOVA, analysis of variance.
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