(Received for publication, August 26, 1996, and in revised form, November 29, 1996)
From the Departments of Pediatrics and
§ Molecular Pharmacology, Diabetes and Metabolic Diseases
Research Program, University Medical Center, SUNY/Stony Brook, Stony
Brook, New York 11794-8651
Transgenic BDF-1 mice harboring an inducible,
tissue-specific transgene for RNA antisense to Gq
provide a model in which to study a loss-of-function mutant of
G
q in vivo. G
q deficiency induced in liver and white adipose tissue at birth produced increased body mass and hyperadiposity within 5 weeks of birth that persisted throughout adult life. G
q-deficient adipocytes display
reduced lipolytic responses, shown to reflect a newly discovered,
1-adrenergic regulation of lipolysis. This
1-adrenergic response via phosphoinositide hydrolysis
and activation of protein kinase C is lacking in the G
q
loss-of-function mutants in vivo and provides a basis for the increased fat accumulation.
Heterotrimeric G-proteins
(G-proteins)1 mediate transmembrane
signaling from a populous group of cell-surface receptors to a lesser
group of effector molecules that includes adenylyl cyclase, phospholipase C, and various ion channels (1). G-proteins have been
shown to regulate complex biological processes, including cellular
differentiation (2, 3), neonatal development (4-6), and oncogenesis
(7). The expression of Go, for example, is highly
localized to the growth cones of developing neurites (2). Suppression
of G
o expression provokes the collapse of developing growth cones (8), whereas expression of constitutively active mutants
of G
o promote increased expression of neurites (9). In
adipogenesis of 3T3 L1 embryonic fibroblasts, G
s acts as
a suppressor (2). Inducers of differentiation stimulate a sharp decline
in G
s levels, and constitutive expression of
G
s blocks induction of differentiation (2).
G
i2 has been shown to regulate the progression of
embryonic stem cells to primitive endoderm (4), acting via
phospholipase C (PLC) and protein kinase C to suppress progression
(10). The morphogen retinoic acid induces primitive endoderm by
stimulating a sharp decline in G
i2 (4). Mimicking the
decline with oligodeoxynucleotides antisense to G
i2
provokes progression in the absence of retinoic acid (4, 10). Study of G-proteins in vivo is a formidable task. The
role of G
i2 in vivo has been studied through
inducible, tissue-specific ablation by antisense RNA (5, 6) and gene
inactivation by homologous recombination (11). Deficiency in
G
i2 leads to a runted phenotype (5, 6, 11), insulin
resistance (12), and for the transgenic mice with the inactivated
G
i2 gene, ulcerative colitis and adenocarcinoma of the
colon (11).
Little is known about the role of G-proteins of the Gq
family in vivo. Two highly homologous members of the
Gq subfamily of G-proteins, Gq and
G
11, can stimulate PLC and are insensitive to pertussis
toxin (13-17). G
11 have been shown to mediate growth in
fibroblasts in response to bradykinin and thrombin (18), hypertrophy in
cultured neonatal ventricular myocytes (19), and transformation in NIH
3T3 cells (20). In the current work, we employ conditional,
tissue-specific expression of RNA antisense to G
q in
transgenic mice to explore the role of this G-protein in
vivo and more specifically in white adipocytes made deficient of
G
q.
[3H]cyclic AMP,
[32P]dCTP, [-32P]ATP,
[3H]inositol 1,4,5-trisphosphate (IP3), Gene
Screen Plus, and anti-G
i3 antibodies (EC2) were
purchased from Dupont NEN. All other reagents were purchased from
Sigma or standard suppliers (5).
The B6D2F1 (BDF1) strain of mice was purchased from Taconic Farms Inc. and handled in accordance with the guidelines established by the Institutional Animal Care and Use Committee at the State University of New York at Stony Brook.
Experimental Design of the Antisense RNA StrategyThe
pPCK-ASGq expression vector was constructed as described
below using standard techniques. In order to insert the antisense sequence at the BglII site within the first exon of the
phosphoenolpyruvate carboxykinase (PEPCK) gene, the 7.0-kb PEPCK gene
was subcloned as a 1.0-kb EcoRI/HindIII and a
5.8-kb HindIII/BamHI fragment into the vector
pGEM7Zf(+) (Promega). The vector harboring the 1.0-kb gene fragment was
digested with BglII, and the restriction ends were made
flush using the Klenow fragment. The 39-bp antisense sequence was
obtained within a 235-bp NheI/SstI fragment
excised from the vector pLNC-ASG
i2 (4). The restriction
ends of this fragment were filled-in, ligated with the
BglII-digested gene fragment, and used to transform XL-1
Blue strain of Escherichia coli (Stratagene) under selection
with ampicillin. Plasmids with the 1.2-kb fragment and an insert
oriented to produce antisense RNA were identified by direct DNA
sequencing. The 1.2-kb fragment containing the antisense sequence was
digested with SacII and ClaI (sites present in
the 235-bp sequence harboring the antisense sequence), and a 59-bp
oligomer containing the sense sequence to G
q (
33 to
+3) flanked by restriction enzyme sites for BclI (5
) and
SalI (3
) was ligated via force cloning into the
SacII and ClaI sites. Insertion of the oligomer
was confirmed by restriction digest analysis with BclI and
SalI. The presence of unique restriction sites within the
235-bp fragment facilitates the removal and insertion of different
antisense sequences in a cassette-like fashion. The 1.2-kb fragment
containing the antisense sequence was excised and ligated into the
plasmid harboring the 5.8-kb gene fragment to produce the 7.0-kb
pPCK-ASG
q construct. In addition, the synthesis of
primers complimentary to the flanking ends of the 235-bp insert allows
for discrimination between the pPCK-ASG
q RNA and the
endogenous PEPCK RNA in subsequent reverse transcription-PCR
amplification reactions (see below).
In choosing the antisense RNA target sequence, we had to consider the
large degree of nucleotide identity among the G-protein -subunits
within their respective open reading frame regions. This prompted us to
look for unique target sequences within the 5
- and 3
-untranslated
regions of the G
q mRNA. The 36 nucleotides immediately upstream of and including the translation initiation codon
were chosen to serve as the antisense target sequence. This 39-base
pair sequence (5
-CGCGCCGGCGGGGCTGCAGCGAGGCACTTCGGAAGAATG-3
) did not
show any significant homology with sequences present in the
GenBankTM data base, including G
11 (33%
homology) and G
14 (27% homology).
FTO-2B cells were cultured in
a 5% CO2, 95% O2 chamber and maintained in
Ham's F-12/Dulbecco's modified Eagle's medium (1:1) supplemented
with 10% fetal bovine serum. Cells were cotransfected with a plasmid
that would allow for neomycin selection of positive transfectant
clones. The control vector or pPCK-ASGq construct was
added in 5-fold excess relative to the plasmid containing the
selectable marker. Transfection was carried out using the Lipofectin
reagent (Life Technologies, Inc.) according to the manufacturer's
protocol.
Total RNA was extracted as described previously (4). One microgram of total RNA was reverse transcribed using a pPCK-ASGaq-specific downstream primer and then PCR-amplified in the presence of both the upstream and downstream primer set according to the manufacturer's protocol (Perkin-Elmer). The sequences for the upstream and downstream primers were dCGTTTAGTGAACCGTCAGA and dAGGTGGGGTCTTTCATTCCC, respectively.
Production of Transgenic MiceTransgenic lines of mice were
produced at the Transgenic Mouse Facility at SUNY Stony Brook using
standard techniques (5, 6). Briefly the pPCK-ASGq
construct was excised free of vector sequences and purified prior to
microinjection into single-cell preimplantation embryos. Microinjected
embryos were then transferred to pseudopregnant females. Offspring
carrying the transgene were identified by PCR amplification and
subsequent Southern analysis using a pPCK-ASG
q-specific
probe uniformly labeled with [32P]dCTP (5). Five separate
founder lines were identified by Southern analysis and bred over 10 generations (5).
White adipocytes were isolated from epididymal and parametrial fat pads by collagenase digestion, as described previously (5). Briefly, 0.5-1.0 g of adipose tissue was excised from male and female mice, weighed, and added to an equal volume of Krebs-Ringer phosphate buffer (KRP) containing 3% bovine serum albumin (KRP/BSA), prewarmed to 37 °C. The tissue was digested for 1 h using collagenase (1 mg/ml) at 37 °C in an orbital, shaking water bath. The isolated adipocytes were washed twice with the KRP/BSA buffer and then resuspended to a final volume to achieve 62.5 mg of wet weight of packed adipocytes/ml in the same buffer. The KRP/BSA buffer was supplemented with adenosine deaminase at a concentration of 0.5 unit/ml.
Cyclic AMP Accumulation and LipolysisBriefly, collagenase-digested white fat cells from epididymal and epoophoronal pads were incubated at 37 °C in KRP buffer supplemented with 3% bovine serum albumin and adenosine deaminase (0.5 unit/ml) for 30 min in the absence or presence of the drugs indicated. For lipolysis determinations, the assays were terminated with 0.65 N HClO4. Samples were deproteinized and neutralized with KOH/KCl/imidazole (2.6/0.52/0.52 M, respectively) and the glycerol content determined by measuring the reduction of NAD+ to NADH in a coupled assay. NADH production was assayed using a microplate fluorometer set to an excitation wavelength of 360 nm and an emission wavelength detection of 460 nm. The mass of glycerol per sample was extrapolated from a standard curve of stock glycerol. Cyclic AMP accumulation was measured using a competitive binding assay. Briefly, 80 µl of fat cells (~5 mg/tube) were treated with various agents for 6 min at 37 °C. The reaction was stopped by the addition of HCl (0.1 N final) and boiling for 1 min. The samples were neutralized with NaOH and assayed for cyclic AMP content. Cyclic AMP accumulation was measured in adipocytes stimulated with either phenylephrine, epinephrine, norepinephrine, or isoproterenol The data are expressed as the mean values in picomoles of cyclic AMP (±S.E.) per million cells from three independent trials, each performed in triplicate.
IP3 and 1,2-sn-Diacyglycerol (DAG) AccumulationCells were incubated with the indicated agents and the IP3 measured as described previously (21). DAG was determined using the DAG kinase assay (10). The data are expressed as the mean values in nanomoles (±S.E.) per million cells from three independent trials, each performed in triplicate.
G-protein Immunoblot AnalysisMembrane fractions were
prepared from rat hepatoma FTO-2B cells, as described previously (5).
Cell membranes were prepared from adipose tissue of transgenic mice and
their control littermates (5). Aliquots of cell membrane were subjected
to SDS-polyacrylamide (10% acrylamide) gel electrophoresis, and the
separated proteins were transferred to nitrocellulose blots. The blots
of the membrane proteins were probed with anti-peptide antibodies
specific for Gq (antibodies E973 and E976),
G
i2 (antibody CM112), G
s (antibody CM129), G
2 (antibody CM162), G
11
(antibody E976), or G
i3 (antibody EC2), and the immune
complexes were made visible by staining with a calf alkaline
phosphatase-conjugated, goat anti-rabbit IgG second antibody (4, 5).
The "CM" antibodies were prepared by our laboratory (4, 5).
Defining the role of a specific G-protein subunit, like
Gq, in vivo is a formidable task. We adopted
the strategy of conditional, antisense RNA to ablate
G
q in vivo in a tissue-specific manner, creating loss-of-function mutants in adipose and liver, prominent sites
of G
q expression. The degree of nucleotide identity
among the G-protein
-subunits within the open reading frames
dictated selection of the 5
-untranslated region immediately upstream
and including the ATG initiator codon (
33 to +3) as the antisense RNA
sequence targeting Gaq (Fig. 1). This region
is unique with respect to sequences with the GenBankTM data
base and does not share significant homology with other G-protein
-subunits (Fig. 1A), including other members of the Gq family, G
11 (33% homology with respect
to G
q) and G
14 (27% homology with
respect to G
q). A double-stranded oligodeoxynucleotide fragment antisense to G
q was inserted into
BclI and SalI sites of the pPCK-AS vector (Fig.
1B), an inducible expression vector driven by the promoter
of the PEPCK gene (5, 6). Screening of FTO-2B hepatoma cells stably
transfected with pPCK-ASG
q was performed from day 0 to
day 12 following induction of the promoter with the chlorophenylthio
analogue of cyclic AMP (CPT-cyclic AMP, 25 µM).
Immunoblots reveal that levels of G
i2,
G
s, and G
2 were unaffected by induction
of pPCK-ASG
q, while staining with an antibody to
G
q displayed a 54% loss by day 6, and 86% loss of G
q by day 9 following induction (Fig. 1C). By
day 12 of the induction with CPT-cyclic AMP, G
q was not
detectable in the immunoblots of FTO-2B cell membranes. Induction of
the pPCK-SG
q vector, harboring the sense as compared to
antisense sequence for G
q, resulted in a null phenotype,
i.e. G
q expression was normal.
G
q activates PLC-
in the liver (12-16) and
suppression of G
q in FTO-2B hepatoma cells reduced basal
PLC activity from 2.7 ± 0.6 to 1.0 ± 0.3 (p
0.05 for difference) and abolished PLC stimulation in response to
either 10 nM angiotensin II (0.9 ± 0.3) or 10 µM norepinephrine (0.95 ± 0.2), as determined by
mass assay of intracellular IP3 accumulation at 30 s
following hormonal stimulation (n = 5, pmol of
IP3 accumulation/µg of cellular protein).
Gq expression is suppressed in
hepatoma cell stably transfected with pPCK-ASG
q and in
mice harboring the pPCK-ASG
q transgene. Comparison
of the 5
-untranslated region from Gaq (nucleotides
33 to
+3) with G
14 and G
11, additional members
of the Gq family (panel A). The
pPCK-ASG
q construct for inducible expression of RNA
antisense to G
q (panel B). The 36-nucleotide
sequence upstream of and including the translation initiation codon was
inserted into the first exon of the rat phosphoenolpyruvate
carboxykinase gene (PCK) to provide a 2.8-kb hybrid
pPCK-ASG
q antisense RNA, driven by a promoter which is
silent in utero and activated at birth. Crude membranes (0.2 mg of protein/SDS-polyacrylamide gel electrophoresis lane) were
prepared from rat hepatoma FTO-2B cells that were stably transfected
with the pPCK-ASG
q construct and induced with CPT-cyclic
AMP for 0, 6, 9, and 12 days, subjected to SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose blots, and probed
with rabbit polyclonal antisera specific for the G-protein subunits
indicated (5, 6). Immunecomplexes were made visible with goat
anti-rabbit IgG coupled to calf alkaline phosphatase and colorimetric
development (panel C). Crude membranes were prepared from
epididymal and epoophoronal white fat and liver (panel D)
and brain and lung (panel E) tissues obtained from
24-week-old control (C) and transgenic (T) mice.
Samples (15 µg of protein/lane) were subjected to SDS-polyacrylamide
gel electrophoresis on a mini-gel apparatus and transferred to
nitrocellulose for immunoblot analysis of various G-protein subunits,
as described earlier (5, 6). For immunoblotting, the sample loading was
limited to 15 µg/lane, within the range established for linearity
between sample loading and quantification of immunostaining (not
shown). Quantification of the blots revealed no significant change in
the G-protein subunits tested between control and transgenic mouse
tissues, with the exception of the loss of G
q in liver
and fat tissues. Expression of G
s was normal in liver
and fat, although reduced (<15%) occasionally in fat, but not liver,
of some transgenic mice (not shown). Scanning densitometry values for
immunoblots of fat tissue from control and transgenic mice,
respectively, were as follows: G
q, 0.26, 0.01;
G
s, 0.92, 0.88; G
, 0.31, 0.33; and
G
i2, 0.72, 0.75 arbitrary OD units. Scanning
densitometry values for immunoblots of liver tissue from control and
transgenic mice, respectively, were as follows: G
q,
0.51; 0.02; G
s, 0.68, 0.64; G
, 0.27, 0.25; and G
i2, 0.22, 0.25 arbitrary OD units. Scanning
densitometry of immunoblots from brain and lung revealed no significant
differences in the values obtained with tissues from transgenic as
compared to control mice (not shown). The antibodies employed for
staining of immunoblots for specific G-proteins subunits were as
follows: E973 for G
q; CM112 for G
i2;
CM129 for G
s; E976 for G
11; and CM162 for
G
2.
The pPCK-ASGq construct was excised as a 7.0-kb
EcoRI-BamHI fragment, microinjected into single
cell, preimplantation embryos, and the microinjected embryos were
transferred into pseudopregnant recipients. BDF1 mice harboring the
transgene were identified by PCR of tail DNA. Five independent founders
were identified from two rounds of microinjection and implantation.
Five separate founder lines have been propagated for more than 10 generations. Immunoblots of crude membranes from fat, liver, brain, and
lung subjected to SDS-PAGE and stained with a
G
q-specific antiserum reveal the near absence of
G
q in the tissues, fat and liver, targeted by the
transgene (Fig. 1D). Immunoblots of brain and lung, tissues
not targeted by the PEPCK vector, displayed normal levels of
G
q (Fig. 1E). Expression of
G
i2, G
2, and G
11 (not shown) were not significantly altered in the transgenic mice.
Necropsy and histology of the transgenic mice were performed. Prominent
was the increase in body weight observed in the mice harboring the
pPCK-ASGq transgene (Fig. 2A).
By 5 weeks of age, the transgenic mice were >135% of the body weight
of their control littermates, for both male and female mice alike (Fig.
2B). Progeny of the five founder lines harboring the
transgene all display the increased body weight (not shown). The fat
mass at 4 weeks after birth increased by 50% in the transgenic mice
(Fig. 2C). At 8 weeks of age, the white epididymal and
epoophoronal fat mass of the transgenic mice was 1.75-fold greater than
that of the control mice. Segregated by gender for males, white fat
mass (mg) was 215 ± 5 and 333 ± 8 (n = 5, p
0.05) for 12-week-old control and transgenic mice,
respectively. For females, white fat mass was 160 ± 10 mg and
303 ± 8 mg (n = 6, p
0.05) for
12-week-old control and transgenic mice, respectively. By 24 weeks, the
transgenic mice displayed a 1.4-fold increase in fat mass, and the
percentage of whole body weight as fat mass was 2.3 ± 0.2 as
compared to 1.1 ± 0.3 (n = 6) for control mice
(Fig. 2C). Total body protein and nasal-anal length were
unaffected by the presence of the transgene over this same range in age
(not shown). Equally notable was the dramatic increase in adiposity,
i.e. fat cell number, that occurred in the transgenic mice
lacking G
q expression in adipose tissue (Fig.
2D).
To assess the effects of Gq deficiency on cell
signaling, we investigated the adipocytes isolated from transgenic mice
and their control littermates. PLC-
signaling by loss-of-function G
q-deficient white adipocytes was virtually abolished,
i.e. IP3 and DAG accumulation in response to
norepinephrine, vasopressin, phenylephrine, or bradykinin (all hormones
that activate PLC) was markedly attenuated (Fig. 3,
A and B, respectively). Suppression of
G
q in adipocytes of the pPCK-ASG
q mice
and the stably transfected hepatoma cells abolished PLC activation by a
variety of hormones (Figs. 1 and 3). This loss of signaling in
G
q deficiency occurs, although expression of the
G
11 subunit was found to be normal (not shown). Both
G
q and G
11 are expressed in a number of
tissues (12-14), including fat and liver. The observations from the
present study suggest that G
q and G
11 may
not be redundant with respect to PLC activation in vivo.
Since PLC activation and accumulation of either IP3 or DAG
have not been implicated in controlling lipolysis, the pharmacology of
the lipolytic response observed in the Gq-deficient
adipocytes came as a great surprise (Fig. 3C). The lipolytic
response to a mixed
- and
-adrenergic agonist norepinephrine was
blunted in the G
q-deficient adipocytes. Lipolysis in
response to the
-adrenergic agonist isoprenaline was impaired,
whereas the response to the
1-adrenergic agonist
phenylephrine was abolished in the loss-of-function mutant cells. These
results were unexpected, since neither a direct role of
G
q in activating adenylyl cyclase nor the existence of a
prominent
1-adrenergic stimulation of lipolysis have
been reported. Analysis of cyclic AMP accumulation provided direct
proof linking loss of G
q to impaired lipolysis in
response to
-adrenergic stimulation (acting via adenylyl cyclase) as
well as to
1-adrenergic stimulation (acting via PLC).
Forskolin (10 µM)-stimulated cyclic AMP accumulation, in
contrast, was actually elevated in the G
q-deficient as
compared to control adipocytes (125 ± 15 and 160 ± 5 pmol/106 cells, respectively). Forskolin (10 µM)-stimulated lipolysis was equivalent in transgenic and
control mice (14.1 ± 2.9 and 13.9 ± 0.8 µmol of glycerol
release/106 cells, respectively), as were the abundance of
-adrenergic receptors (140 ± 4 and 133 ± 9 fmol/mg of
protein, respectively) in crude adipocyte membranes and the amounts of
cyclic AMP phosphodiesterase activity (1.33 ± 0.02 and 1.32 ± 0.09 pmol/min/mg of protein, respectively) in extracts of whole fat
pads.
In the Gq-deficient cells, the impaired lipolytic
response stimulated by norepinephrine was sensitive to the strict
-adrenergic antagonist propranolol, reflecting a residual
-adrenergic, cyclic AMP-mediated response (Fig.
4A). Adipocytes from control mice display
sensitivity to both propranolol and the
1-adrenergic antagonist prazosin. The former reflects the
-adrenergic response acting via cyclic AMP, while the latter reflects this newly discovered
1-adrenergic response first detected through its loss in
the G
q-deficient cells. Vasopressin (1 µM), which activates PLC, also stimulated lipolysis in
adipocytes from control mice 1.8-fold over basal. The
1-adrenergic stimulation of lipolysis was abolished by
prazosin, but not by propranolol (Fig. 4B). The
loss-of-function G
q-deficient cells, in contrast, have
essentially lost the lipolytic response to phenylephrine
stimulation.
The pharmacology of the adrenergic lipolytic
response reveals the existence of an 1-adrenergic
stimulatory pathway, absent in the G
q-deficient
loss-of-function mutants. White adipocytes were isolated from
transgenic mice and their control littermates for study of the lipolytic response to adrenergic agonists. The lipolytic response was measured as described in the legend to Fig. 3.
Stimulation of lipolysis by either 10 µM norepinephrine (NOR, panel A) or 10 µM
phenylephrine (PHEN, panels B and C)
was analyzed in the absence and presence of either the
-adrenergic antagonist propranolol (PROP, 10 µM) or the
1-adrenergic antagonist prazosin (PRAZ, 1 µM). Inhibitors of protein kinase A (KT,
KT5702, 1 µM) and protein kinase C (BIS,
bis-indolylmaleimide, 1 µM; CAL, calphostin C,
100 nM) were examined for their ability to block the
1-adrenergic stimulation of lipolysis in adipocytes from transgenic mice and their control littermates (panel C). The
results are mean values ± S.E. from three to five separate
experiments for each. An asterisk denotes statistical
significance with p
0.05 for the differences from
mean basal values obtained with adipocytes isolated from both
transgenic (G
q-deficient) and control mice.
Although Gq is not known to regulate adenylyl cyclase
directly, the loss-of-function G
q mutants displayed
impaired
-adrenergic stimulation of cyclic AMP accumulation and
lipolysis. G
q, acting via PLC to promote IP3
and DAG accumulation, may augment the cyclic AMP response indirectly,
perhaps via effects on calcium- or protein kinase C-sensitive forms of
adenylyl cyclase (22-25). We tested the role of protein kinase C using
bis-indolylmaleimide and calphostin C, selective inhibitors of protein
kinase C (Fig. 4C). Both calphostin C (100 nM)
and bis-indolylmaleimide (1 µM) abolished the
1-adrenergic stimulation of lipolysis, whereas the
protein kinase A inhibitor KT-5720 (1 µM) was without
effect. At 100 nM, bis-indolylmaleimide effectively blocked
phenylephrine (10 µM)-stimulated lipolysis in adipocytes
from control mice; glycerol release (µmol/106 cells), in
response to this
1-adrenergic agonist, declined from 4.7 to 1.2 in the absence versus presence of this protein kinase C inhibitor. The KI for nonselective inhibition of
protein kinase A by bis-indolylmaleimide is >2 µM (26).
Since the protein kinase A inhibitor KT-5720 itself was without
effect, nonselective effects of protein kinase C inhibitors, if they
indeed occurred at these lower concentrations, would be irrelevant.
Measurement of protein kinase C activity in DEAE-cellulose-purified
homogenates of cells challenged with and without norepinephrine was
performed using adipocytes from the control and transgenic mice (Fig.
5). In adipocytes from control mice, norepinephrine stimulates protein kinase C activity, an action blocked by the addition
of bis-indolylmaleimide. Suppression of Gq in adipocytes of the pPCK-ASG
q mice results in a frank reduction in
protein kinase C activity in the basal state and a loss of
norepinephrine-induced activation of protein kinase C. Total protein
kinase activities for adipocytes from control and transgenic mice are
equivalent, 310 ± 20 and 315 ± 18 pmol/min/million cells,
respectively. Thus,
1-adrenergic control of lipolysis is
shown to be mediated via protein kinase C, a pathway revealed by its
absence in the G
q-deficient state.
The absence of Gq resulted in increased fat accumulation
and hyperadiposity, observed within 5 weeks of age and sustained through adult life. Obesity has been reported in transgenic mice after
genetic ablation of brown adipose tissue (27), supporting the role of
this specialized tissue in preventing obesity (28). The
pPCK-ASG
q transgene was not expressed in brown adipose
tissue (not shown). Expression of G
q, the
uncoupling protein UCP, and the mRNAs for both were
equivalent in brown adipose tissue from transgenic and control mice
(not shown), suggesting no involvement of brown adipose tissue in
enhanced fat accumulation by the pPCK-ASG
q-expressing mice.
The absence of Gq abolished an important stimulatory
control of lipolysis, apparently predisposing the mice to accumulation of fat. Recently, G-proteins have been shown to play prominent roles in
differentiation (2, 3) and neonatal growth (4-6). For progression of
F9 teratocarcinoma cells to primitive endoderm (4) and for development
of nerve growth cones (3), G-proteins appear to be acting directly or
indirectly via protein kinase C (10, 29). In the present study we
demonstrate the key role of PLC and protein kinase C in adipocyte
signaling in the mature cells. The basis for the hyperadiposity in the
cells deficient in G
q, however, remains to be
established, but may reflect a critical role of G
q in
controlling adipogenic conversion in vivo.
All authors contributed equally to work reported herein.
We thank Dr. John H. Exton (HHMI, Vanderbilt University, Nashville, TN) for the generous gift of antibodies E973 and E976. We thank Dr. Jean Himms-Hagen (University of Ottawa, Ontario, Canada) for the generous gift of antibodies to UCP.