Overexpression of 1-Acyl-Glycerol-3-Phosphate Acyltransferase-
Enhances Lipid Storage in Cellular Models of Adipose Tissue and Skeletal Muscle
Hong Ruan, and
Henry J. Pownall
From the Section of Atherosclerosis and Lipoprotein Research (H.J.P.,
H.R.), Department of Medicine, Baylor College of Medicine; and The Methodist
Hospital (H.J.P.), Houston, Texas.
Address correspondence and reprint requests to Henry J. Pownall, PhD,
Department of Medicine, MS A-601, Baylor College of Medicine, 6565 Fannin St.,
Houston, TX 77030. E-mail:
hpownall{at}bcm.tmc.edu
.
 |
ABSTRACT
|
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Plasma nonesterified fatty acids (NEFA) at elevated concentrations
antagonize insulin action and thus may play a critical role in the development
of insulin resistance in type 2 diabetes. Plasma NEFA and glucose
concentrations are regulated, in part, by their uptake into peripheral
tissues. Cellular energy uptake can be increased by enhancing either energy
transport or metabolism. The effects of overexpression of
1-acylglycerol-3-phosphate acyltransferase (AGAT)-
, which catalyzes the
second step in triglyceride formation from glycerol-3-phosphate, was studied
in 3T3-L1 adipocytes and C2C12 myotubes. In myotubes, overexpression of
AGAT-
did not affect total [14C]glucose uptake in the
presence or absence of insulin, whereas insulin-stimulated
[14C]glucose conversion to cellular lipids increased significantly
(33%, P = 0.004) with a concomitant decrease (-30%, P =
0.005) in glycogen formation. [3H]oleic acid (OA) uptake in
AGAT-overexpressing myotubes increased 34% (P = 0.027) upon insulin
stimulation. AGAT-
overexpression in adipocytes increased basal (130%,
P = 0.04) and insulin-stimulated (27%, P = 0.01)
[3H]OA uptake, increased insulin-stimulated glucose uptake (56%,
P = 0.04) and conversion to cellular lipids (85%, P =
0.007), and suppressed basal (-44%, P = 0.01) and
isoproterenol-stimulated OA release (-45%, P = 0.03) but not glycerol
release. Our data indicate that an increase in metabolic flow to triglyceride
synthesis can inhibit NEFA release, increase NEFA uptake, and promote
insulin-mediated glucose utilization in 3T3-L1 adipocytes. In myotubes,
however, AGAT-
overexpression does not increase basal cellular energy
uptake, but can enhance NEFA uptake and divert glucose from glycogen synthesis
to lipogenesis upon insulin stimulation.
 |
INTRODUCTION
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Type 2 diabetes is characterized, in part, by excess energy as glucose and
nonesterified fatty acids (NEFA) within the plasma compartment. Moreover, an
elevated plasma NEFA concentration has been increasingly recognized as a
systemic mediator of insulin resistance in type 2 diabetes
(1,2,3,4,5).
Excess plasma NEFA can inhibit insulin-stimulated glucose utilization in
muscle
(1,2,3,4)
and promote hepatic production of glucose
(4,5,6)
and VLDL triglyceride (TG)
(7,8).
Acute systemic administration of NEFA inhibits glucose disposal in muscle in a
dose-dependent fashion (4) and
increases hepatic glucose output
(5,6).
Reduction of plasma NEFA concentration improves glucose utilization
(9,10,11),
enhances the suppression of hepatic glucose production by insulin
(12), and reduces
hyperinsulinemia in patients with type 2 diabetes
(13). Thus, plasma NEFA
elevation may be mechanistically linked to the cluster of metabolic
abnormalities seen in type 2 diabetes, including hyperglycemia,
hyperinsulinemia, and dyslipidemia.
Skeletal muscle and adipose tissue are major sites of energy utilization.
Most of the glucose and NEFA taken up by resting muscle is converted to
glycogen (14) and TG
(15). Because of its mass,
skeletal muscle is a significant storage site for excess plasma NEFA in
patients with type 2 diabetes
(16) and in animal models of
the disease
(17,18).
However, the accumulation of intramuscular TG may increase lipid oxidation and
decrease glucose uptake and insulin sensitivity
(18,19).
In contrast, increasing NEFA and glucose uptake in adipose tissue could reduce
systemic NEFA availability and would eventually improve insulin sensitivity in
liver and muscle.
Energy uptake can be increased by enhancing either of the proposed
rate-limiting steps of energy utilization, i.e., energy transport across the
plasma membrane or energy metabolism within the cell. Indeed, an increase in
glucose transport
(20,21,22)
or metabolism
(23,24)
promotes glucose utilization in cultured cells and transgenic animals.
Moreover, glucose metabolism appears to be rate limiting under conditions of
enhanced glucose transport
(22,25,26,27,28,29).
Less is known about the regulation of NEFA uptake. Overexpression of
long-chain fatty acid transport protein (FATP) increases NEFA uptake in 3T3-L1
fibroblasts (30). On the other
hand, an increase in cytoplasmic fatty acyl-CoA synthase (FACS) activity
without a change in FATP is sufficient to increase NEFA uptake
(30), indicating that NEFA
transport is not rate limiting and that intracellular NEFA metabolism can
drive NEFA uptake. Because insulin promotes energy uptake in adipose tissue
and muscle, and because the ultimate effect of insulin is to enhance energy
storage, we hypothesized that an increase in energy flow to TG synthesis would
enhance cellular uptake of NEFA and, under certain conditions, glucose as
well.
Four enzymes are involved in TG synthesis from glycerol-3-phosphate and
fatty acyl-CoA (31). The
second step in TG formation is catalyzed by 1-acylglycerol-3-phosphate
acyl-transferase (AGAT), which converts 1-acylglycerol-3-phosphate
(lysophosphatidic acid) (LPA) to 1,2-diacylglycerol-3-phosphate (phosphatidic
acid) (PA). AGAT occurs as
and ß isoforms
(32), which are expressed at
high levels in adipose tissue and skeletal muscle (H.R. and H.J.P.,
unpublished data). To study the effects of AGAT-
on cellular TG
synthesis and uptake of NEFA and glucose, we established stable 3T3-L1 and
C2C12 cell lines representing adipose tissue and skeletal muscle,
respectively, which overexpress AGAT-
. Herein, we report those
effects.
 |
RESEARCH DESIGN AND METHODS
|
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Cell culture. 3T3-L1 cells were purchased from the American Type
Culture Collection (Rockville, MD), maintained as fibroblasts, and
differentiated into adipocytes as previously described
(33). C2C12 myoblasts,
obtained from Robert J. Schwartz, PhD, were maintained in Dulbecco's minimum
essential medium (DMEM) supplemented with 10% fetal calf serum. After
confluence, the culture medium was changed to DMEM with 2% horse serum to
initiate myogenic differentiation. Before each assay, both 3T3-L1 adipocytes
and C2C12 myotubes were serum starved for 3 h in Krebs-Ringer bicarbonate
buffer solution with 25 mmol/l Krebs-Ringer bicarbonate HEPES buffer (KRBH)
(pH 7.4) containing 100 µmol/l bovine serum albumin (BSA) (Sigma, St.
Louis, MO) essentially fatty acid free, and 5 mmol/l glucose.
Stable cell lines. A 2,083-bp cDNA clone encoding human AGAT was
isolated from a human adipocyte cDNA library (Clontech, Palo Alto, CA) using a
human infant brain expressed sequence tag similar to yeast AGAT (GenBank no.
T77083; Genome Systems, St. Louis, MO) as a hybridization probe. The cDNA
clone contains an 852-bp open reading frame encoding a protein of 283 amino
acids with a calculated molecular mass of 31.7 kDa. Membrane preparations from
AGAT-deficient Escherichia coli strain JC-201
(34) transformed with
full-length AGAT cDNA exhibited AGAT activity (H.R. and H.J.P., unpublished
data). This AGAT was identical to the
isoform described by West et al.
(32).
The polymerase chain reaction (PCR) method was used to delete the predicted
NH2-terminal 30-amino acid leader sequence of AGAT-
protein
and to introduce an EcoRI site into the upstream sequence and a
NotI site into the downstream sequence of AGAT-
. The resulting
PCR fragment was subcloned into the pcDNA3.1/HisC vector (Invitrogen,
Carlsbad, CA); its sequence was verified by DNA sequencing. The recombinant
plasmid, pHis-AGAT, encodes 293 amino acids containing a 40-amino acid
NH2-terminal epitope tag (including six histidines) and has a
calculated mass of 32.3 kDa. This expression construct was transfected into
3T3-L1 fibroblasts and C2C12 myoblasts using SuperFect reagent (Qiagen,
Valencia, CA). After selection in G-418, pools of 40-60 surviving colonies
were expanded for study. Control cell lines were transfected with pcDNA/HisC
vector without the insert, followed by G-418 selection. Cell line expression
of AGAT-
was confirmed by Western blot analysis and immunofluorescence
microscopy using a monoclonal anti-His6 antibody (Clontech). The
transfection and selection were repeated, and the independent G-418-resistant
clones were amplified and verified for AGAT expression by
reverse-transcriptase-PCR and/or Western blot analysis. Phenotypes from the
two selections were not distinguishable, and data are representative of the
two selections.
Western blotting. Either 30 or 60 µg of total cellular protein
was separated by 12% SDS-PAGE and electroblotted onto a nitrocellulose
membrane (Amersham, Arlington Heights, IL). The filter was incubated for 1 h
at room temperature with monoclonal anti-His6 antibody (1:5,000;
Clontech), washed, and incubated with horseradish peroxidaseconjugated
secondary antibody. Bound antibodies were detected by using the enhanced
chemiluminescence Western blotting analysis system (Amersham).
Immunofluorescence microscopy. Cells were grown on cover slips,
fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, washed
with phosphate-buffered saline (PBS), blocked with 1% normal goat serum in
PBS, and incubated for 1 h at room temperature with monoclonal
anti-His6 antibodies (1:500; Clontech). Cells were washed with PBS
and incubated with fluorescein isothiocyanate (FITC) or rhodamine-conjugated
goat anti-mouse IgG (Pierce, Rockford, IL). Cells were treated with
4,6-diamidino-2-phenylindole as a counterstain, washed extensively with PBS,
and examined using a Zeiss Axiophot fluorescence microscope.
[3H]oleic acid uptake and incorporation into cellular
lipids. After serum deprivation, cells in six-well dishes were incubated
for 1 h in KRBH: 100 µmol/l BSA:5 mmol/l glucose with or without 174 nmol/l
insulin. The assay was initiated by addition of 100 µmol/l
[3H]oleic acid (OA) (5 µCi per well). At various times
(t), aliquots of medium were counted in duplicate, and the uptake was
calculated as (dpmt=0 -
dpmt)/dpmt=0 x 100%, where dpm
is the number of disintegrations per min per aliquot. After 90 min, uptake was
terminated by exhaustive washing with ice-cold PBS. Cells were lysed, and an
aliquot was used to determine cell-associated radioactivity by liquid
scintillation counting. Total cellular lipids were extracted according to a
modified Folch method (35),
dried under a stream of nitrogen, dissolved in CHCl3:MeOH (2:1),
mixed with lipid standards for TG, diglyceride (DG), LPA, and PA, and loaded
onto two thin-layer chromatography (TLC) plates. One plate was developed in
CHCl3:MeOH:H2O (65:25:4), which separates polar lipids,
and the other in hexane:diethyl ether:acetic acid (80:20:2), which separates
neutral lipids. Individual lipid spots were visualized by exposing the dried
TLC plates to iodine vapor and/or a phosphor screen (Bio-Rad, Hercules, CA),
scraped from the plate, and dissolved in scintillation fluid, and
radioactivity was determined by counting.
Lipid and glycogen synthesis. After serum starvation, cells in
six-well dishes were incubated for 15 min in KRBH/BSA without glucose. Insulin
was added for an additional 15 min. The assay was started by adding 5 mmol/l
[14C]glucose (1 µCi per well) to the culture medium, followed by
incubation at 37°C for 1 h. Uptake was terminated by washing the cells
three times with ice-cold PBS containing 10 mmol/l glucose. Cells were lysed
using 1 ml RIPA (150 mmol/l NaCl:1% NP-40:0.5% deoxycholate:0.l% SDS, 50
mmol/l Tris pH 8.0) per well. Cell-associated radioactivity in 50 µl lysate
was determined by liquid scintillation counting. To determine lipid-associated
radioactivity, lipids were extracted from 450 µl cell lysate
(35) and counted. Another
aliquot of cell lysate (450 µl) was used to measure
[14C]glycogen formation
(36).
Lipolysis assay. AGAT-or mock-transfected 3T3-L1 adipocytes grown in
six-well dishes were rinsed with PBS, incubated for 3 h with 600 µmol/l OA
and 300 µmol/l BSA, and washed with DMEM/300 µmol/l BSA three times.
DMEM:600 µmol/l BSA:25 mmol/l HEPES (500 µl) was added to the cells,
which were incubated for 30 min with or without 10 µmol/l isoproterenol. At
the end of the incubation, the medium was collected, concentrated, and assayed
with kits for glycerol (Sigma) and NEFA (Wako Chemicals, Richmond, VA).
AGAT activity in cell extracts. Cell extracts were prepared as
previously described (37), and
total protein was measured using a kit (Bio-Rad). An aliquot of cell extract
(7-10 µg) was added to a reaction mixture containing 50 µmol/l LPA
(oleoly-sn-glycero-3-phosphate) and 50 µmol/l
[14C]oleoyl-CoA (Amersham), followed by incubation for 3-6 min at
30°C. The reaction was terminated by spotting an aliquot onto a silica gel
60 TLC plate, which was developed in chloroform:methanol:water (65:25:4).
Production of radiolabeled lipids was quantified as described above.
Statistical methods. Comparisons were performed using a two-tailed
Student's t test. P
0.05 was considered significant.
Data are means ± SE.
 |
RESULTS
|
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Stable cell lines overexpressing human AGAT-
. Stable 3T3-L1
and C2C12 cell lines expressing His6-tagged human AGAT-
(AGAT-) or containing vector pcDNA3.1/HisC alone (mock-) were generated.
Cellular localization of the His6-AGAT fusion protein, which has an
apparent molecular mass of 34 kDa (Fig.
1), was examined in 3T3-L1 fibroblasts and adipocytes and in C2C12
myoblasts and myotubes incubated with anti-His6 antibody.
Immunofluorescence microscopy showed distinct cytoplasmic staining in all cell
types (Fig. 2, left panels);
staining did not occur in mock-transfected cells
(Fig. 2, right panels).
Overexpression of AGAT-
did not significantly alter cell morphology,
cell proliferation rate, or differentiation of fibroblasts and myoblasts into
adipocytes and myotubes.
Cell extracts of AGAT- and mock-3T3-L1 adipocytes or AGAT- and mock-C2C12
myotubes were assayed for the conversion of [14C]oleoyl-CoA to
[14C]PA. The endogenous AGAT activity in 3T3-L1 adipocyte extracts
was nearly an order of magnitude higher than that in C2C12 myotube extracts
(Table 1). [14C]PA
formation in AGAT-C2C12 myotubes was 150% of that of the mock-transfected
myotubes (P < 0.02). Measures of [14C]PA and
[14C]phosphatidylethanolamine production in AGAT-overexpressing
adipocyte extracts were 123% (P < 0.04) and 131% (P <
0.02) of those of mock-transfected adipocyte extracts.
Effects of AGAT-
overexpression on [3H]OA and
[14C]glucose metabolism in 3T3-L1 adipocytes. [3H]OA
or [14C]glucose uptake by 3T3-L1 adipocytes and C2C12 myotubes was
measured as a function of insulin concentration (data not shown); the maximal
[3H]OA uptake (>90%) by 3T3-L1 adipocytes occurred at 10 nmol/l
of insulin, and was not significantly different between 10 nmol/l and 1
µmol/l of insulin. In C2C12 myotubes, the maximal [14C]glucose
uptake observed was
174 nmol/l. When called for, insulin was used at 174
nmol/l.
[3H]OA uptake in the absence and presence of insulin was
measured in AGAT-L1 and mock-L1 adipocytes. At 90 min, insulin treatment
enhanced [3H]OA uptake in AGAT-L1 cells (65%, P = 0.04)
and mock-L1 cells (170%, P = 0.004), and under both conditions,
[3H]OA uptake was increased by AGAT-
overexpression (by 27%
with insulin treatment and by 130% without insulin treatment)
(Fig. 3).
Total cellular lipids were extracted at the end of the 90-min incubation
and separated by TLC (Fig.
4A). Radioactivity associated with LPA, PA, DG, and TG
was quantified, and the incorporation of [3H]OA into glycerolipids
was assessed. There were no significant differences in cellular
[3H]LPA or [3H]DG content in AGAT-L1 and mock-L1
adipocytes in the presence or absence of insulin
(Fig. 4B). Insulin
increased [3H]PA in both AGAT-L1 and mock-L1 adipocytes (40 and
107%); AGAT-
overexpression increased [3H]PA under both
basal and insulin-stimulated conditions (96 and 33%). Insulin increased
[3H]TG content in mock-L1 adipocytes (181%), but not in AGAT-L1
adipocytes; AGAT-
overexpression increased [3H]TG, but only
under basal conditions (196%). Under all conditions studied, most
[3H]OA was converted to TG.

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FIG. 4A : Effects of AGAT overexpression and insulin treatment on the lipid
profile of 3T3-L1 adipocytes. Adipocytes were treated as described in
Fig. 3. Cellular lipids were
extracted and analyzed by TLC as described in RESEARCH DESIGN AND METHODS.
Left panel, separation of polar lipids. Right panel, separation of neutral
lipids. MB, mock-transfected cells unexposed to insulin (basal); MI,
mock-transfected cells incubated with insulin; AB, AGAT-transfected cells
unexposed to insulin (basal); AI, AGAT-transfected cells incubated with
insulin.
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FIG. 4B : Effects of AGAT overexpression and insulin treatment on the lipid
profile of 3T3-L1 adipocytes. Spots corresponding to LPA, PA, DG, and TG were
transferred to vials, and the associated radioactivity was measured by liquid
scintillation counting. AGAT, AGAT-transfected 3T3-L1 adipocytes; mock,
mock-transfected 3T3-L1 adipocytes. Data are means ± SE of two
experiments. Differences for which P values are not shown are not
statistically significant.
|
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Insulin significantly increased total glucose uptake in both AGAT-L1 and
mock-L1 adipocytes (514 and 330%), and AGAT-
overexpression did not
affect basal glucose uptake but significantly increased insulin-stimulated
glucose uptake (56%) (Fig.
5A). These findings accorded with measurements of the
conversion of glucose to cellular lipids, which significantly increased with
insulin exposure in AGAT-L1 and mock-L1 cells (920 and 350%) and with
AGAT-
overexpression under the condition of insulin stimulation (85%)
(Fig. 5B).
Lipolysis and re-esterification. In the lipolysis assay,
isoproterenol exposure increased glycerol release in both AGAT-L1 and mock-L1
adipocytes (37 and 40%) (Fig.
6A) and OA release in mock-L1 cells (120%)
(Fig. 6B). The
increase in OA release with isoproterenol in AGAT-L1 cells was substantial
(105%) but not significant (Fig.
6B). AGAT-
overexpression affected neither basal
nor isoproterenol-mediated glycerol release
(Fig. 6A), but
suppressed OA release under both conditions (-44 and -45%)
(Fig. 6B). In the
absence of NEFA re-esterification, the concentration of NEFA in the medium
would be expected to be three times that of glycerol. Calculation of the
percentage of NEFA relative to glycerol in the medium yielded values
indicating that most of the mobilized NEFAs were re-esterified, a result
enhanced by AGAT-
over-expression
(Table 2).
Effect of AGAT-
overexpression on [14C]glucose and
[3H]OA utilization in C2C12 myotubes. To identify the role of
AGAT-
in the utilization of fuel molecules by skeletal muscle, the
effects of AGAT-
overexpression on [14C]glucose and
[3H]OA uptake in C2C12 myotubes were assessed in the absence and
presence of insulin. Treatment of fully differentiated muscle cells with 174
nmol/l insulin did not significantly affect [3H]OA uptake in either
mock- or AGAT-C2C12 myotubes (Fig.
7). Similar results were observed between 1 and 500 nmol/l insulin
(data not shown). AGAT overexpression increased [3H]OA uptake by
34% (P = 0.027) in the presence of 174 nmol/l insulin, but not in the
absence of insulin (Fig.
7).
Insulin exposure increased [14C]glucose uptake in both
AGAT-C2C12 and mock-C2C12 myotubes (29 and 23%), although AGAT-
overexpression did not alter either basal or insulin-stimulated glucose uptake
(Fig. 8A). In the
presence of insulin, AGAT-
overexpression altered the metabolic fate of
intracellular glucose; glycogen synthesis decreased (-30%)
(Fig. 8B), and glucose
conversion to lipids increased (33%) (Fig.
8C).
 |
DISCUSSION
|
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Insulin stimulates cellular glucose uptake at multiple steps, including
transport across the plasma membrane
(38) and intracellular
metabolism
(39,40).
Although overexpression of GLUT1 in skeletal muscle of transgenic mice
increases cellular glucose uptake and glycogen content, intracellular glucose
also accumulates (22). Thus,
under conditions of increased glucose transport, cellular glucose metabolism
in skeletal muscle may be rate limiting with respect to wholebody glucose
disposal. Similarly, cellular metabolism of NEFA to glycerolipids, especially
TG, might be the primary determinant of the rate of NEFA uptake in adipose
tissue.
Although the enzymes involved in glycerolipid synthesis have been
identified, the role of each enzyme in cellular TG accumulation and energy
uptake is unknown. We established stable 3T3-L1 and C2C12 cell lines
overexpressing human AGAT-
. These cells were used to evaluate the
effects of AGAT-
overexpression on cellular energy uptake and TG
synthesis.
In both the presence and absence of insulin, overexpression of AGAT-
in 3T3-L1 fat cells increased OA uptake and its incorporation into TG (Figs.
3 and
4B). However, OA
uptake was greater in the presence of insulin, suggesting that the coordinate
regulation of other insulin-responsive genes can enhance the effects of
AGAT-
overexpression. These genes might include those for adipocyte
fatty acid binding protein
(41), FACS
(42), glycerol-3-phosphate
acyltransferase (GPAT)
(43,44),
and hormone-sensitive lipase (HSL). On the other hand, in C2C12 myotubes
insulin did not affect OA uptake, and AGAT-
overexpression produced
only modest increases in OA uptake, which was observed only in the presence of
insulin. Because AGAT activity is seven- to ninefold higher in 3T3-L1
adipocytes than in C2C12 myotubes (Table
1), it is possible that adipocytes have a higher expression of
other adipogenic genes, such as FATP, FACS, GPAT, PA phosphohydrolase, and DG
acyltransferase, which are required for the conversion of NEFA to TG. It is
also possible that C2C12 cells lack the mechanism(s) for activating the
adipogenic genes that would support increased TG synthesis and NEFA
uptake.
In adipocytes, intracellular lipolysis is catalyzed by HSL, which liberates
both glycerol and NEFA; this activity is stimulated by isoproterenol. The
cellular release of glycerol, which cannot be directly esterified by
adipocytes
(45,46),
provides an estimate of HSL activity. In contrast, the NEFA liberated by HSL
can be either released into the medium or re-esterified, so that differences
in the amount of NEFA released and the amount expected on the basis of
glycerol release reflect the effects of NEFA re-esterification. Our data
(Table 2 and
Fig. 6) show that the amount of
NEFA released into the medium was low relative to glycerol under all
conditions studied, indicating that most NEFA liberated by HSL are
re-esterified. Under both basal and isoproterenol-stimulated conditions,
AGAT-
overexpression decreased NEFA release. Because AGAT
overexpression increases cellular TG (Fig.
4B), we conclude that most of the mobilized NEFA return
to the intracellular TG pool, and that AGAT-
overexpression increases
esterification of NEFA derived from both intracellular lipolysis
(Table 1 and
Fig. 6) and the extracellular
medium (Fig. 3).
Cellular glucose metabolism was also altered by AGAT-
overexpression, and the alterations were different in 3T3-L1 and C2C12 cells.
In 3T3-L1 fat cells, AGAT-
overexpression increased glucose uptake and
its conversion to lipids, but only with insulin stimulation
(Fig. 5). The absence of an
effect without insulin is likely because of a strict requirement for
insulin-dependent activities in the pathway that converts extracellular
glucose to intracellular 1-acyl-glycerol-3-phosphate or fatty acyl CoA, the
substrates for AGAT. The insulin-dependent activities may include GLUT4,
hexokinase, and the enzymes involved in lipogenesis, such as acetyl-CoA
carboxylase, pyruvate dehydrogenase, and fatty acid synthase
(47). Similarly, in C2C12
cells, AGAT-
overexpression had no effect on basal glucose uptake. In
the presence of insulin, however, AGAT-
overexpression diverted glucose
from glycogen synthesis to lipid synthesis
(Fig. 8B and
C), presumably because of increased entry of
glucose-6-phosphate into the glycolysis pathway generating acetyl-CoA and
glycerol-3-phosphate.
Because AGAT catalyzes the conversion of LPA to PA
(31), AGAT overexpression in
3T3-L1 adipocytes would be expected to increase cellular PA content. However,
in our experiments, that difference was small; the greatest difference was in
cellular TG content. In contrast to the intact cells, most of the product
formed in cell extracts was PA (Table
1), and AGAT overexpression only modestly increased PA and did not
increase TG formation. This finding is consistent with a model in which AGAT
is proximal to the other enzymes of glycerolipid synthesis. Disruption of
cells could separate these enzymes and uncouple their activities.
Consequently, PA accumulates in the extracts, but not in intact AGAT-L1
adipocytes.
In 3T3-L1 adipocytes, AGAT-
overexpression increases NEFA uptake and
decreases NEFA efflux. Increased entry of NEFA into the glycerolipid synthesis
pathway would lower the cytoplasmic NEFA concentration and in turn create a
NEFA concentration gradient across the plasma membrane. This gradient could
drive NEFA influx, which may be diffusive
(48,49)
or involve NEFA transport proteins
(30). In a physiological
context, increased AGAT activity in adipose tissue might decrease plasma NEFA
concentrations. Future studies of this pathway should include tests of AGAT
overexpression in vivo to determine whether it is an attractive target for
pharmacological management of plasma NEFA and lipoprotein concentrations.
 |
ACKNOWLEDGMENTS
|
---|
This work was supported by grants from the National Institutes of Health
(HL-30914 and HL-56865) and the Ethel L. Walker Memorial Research and
Education Fund for Arteriosclerosis. H.R. was a student in The Michael E.
DeBakey Graduate Program in Cardiovascular Sciences.
 |
FOOTNOTES
|
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AGAT, 1-acylglycerol-3-phosphate acyltransferase; BSA, bovine serum
albumin; DG, diglyceride; DMEM, Dulbecco's minimum essential medium; FACS,
fatty acyl-CoA synthase; FATP, fatty acid transport protein; FITC, fluorescein
isothiocyanate; GPAT, glycerol-3-phosphate acyltransferase; HSL,
hormone-sensitive lipase; KRBH, Krebs-Ringer bicarbonate HEPES buffer; LPA,
lysophosphatidic acid; NEFA, nonesterified fatty acids; OA, oleic acid; PA,
phosphatidic acid; PBS, phosphate-buffered saline; PCR, polymerase chain
reaction; TG, triglyceride; TLC, thin-layer chromatography.
Received for publication October 28, 1999
and accepted in revised form October 23, 2000
 |
REFERENCES
|
---|
-
Randle P, Garland P, Hales C, Newsholme E: The glucose fatty-acid
cycle: its role in insulin sensitivity and the metabolic disturbances of
diabetes mellitus. Lancet 1:785
-789, 1963
-
McGarry JD: What if Minkowski had been ageusic?: An alternative
angle on diabetes. Science 258:766
-770, 1992[Medline]
-
Roden M, Price TB, Perseghin G, Petersen KF, Rothman DL, Cline GW,
Shulman GI: Mechanism of free fatty acid-induced insulin resistance in humans.
J Clin Invest 97:2859
-2865, 1996[Abstract/Free Full Text]
-
Boden G, Chen X, Ruiz J, White JV, Rossetti L: Mechanisms of fatty
acid-induced inhibition of glucose uptake. J Clin
Invest 93:2438
-2446, 1994[Medline]
-
Ferrannini E, Barrett EJ, Bevilacqua S, DeFronzo RA: Effect of
fatty acids on glucose production and utilization in man. J Clin
Invest 72:1737
-1747, 1983[Medline]
-
Ruderman NB, Toews CJ, Shafrir E: Role of free fatty acids in
glucose homeostasis. Arch Intern Med123
: 299-313,1969[Medline]
-
Byrne CD, Brindle NP, Wang TW, Hales CN: Interaction of
non-esterified fatty acid and insulin in control of triacylglycerol secretion
by Hep G2 cells. Biochem J 280:99
-104, 1991[Medline]
-
Laws A: Free fatty acids, insulin resistance and lipoprotein
metabolism. Curr Opin Lipidol7
: 172-177,1996[Medline]
-
Reaven GM, Chang H, Ho H, Jeng CY, Hoffman BB: Lowering of plasma
glucose in diabetic rats by antilipolytic agents. Am J
Physiol 254:E23
-E30, 1988[Abstract/Free Full Text]
-
Reaven GM, Chang H, Hoffman BB: Additive hypoglycemic effects of
drugs that modify free-fatty acid metabolism by different mechanisms in rats
with streptozocin-induced diabetes. Diabetes37
: 28-32,1988[Abstract]
-
Balasse EO, Neef MA: Influence of nicotinic acid on the rates of
turnover and oxidation of plasma glucose in man.
Metabolism 22:1193
-1204, 1973[Medline]
-
Saloranta C, Franssila-Kallunki A, Ekstrand A, Taskinen MR, Groop
L: Modulation of hepatic glucose production by non-esterified fatty acids in
type 2 (non-insulin-dependent) diabetes mellitus.
Diabetologia 34:409
-415, 1991[Medline]
-
Boden G, Chen X, Iqbal N: Acute lowering of plasma fatty acids
lowers basal insulin secretion in diabetic and nondiabetic subjects.
Diabetes 47:1609
-1612, 1998[Abstract]
-
Shulman GI, Rothman T, Jue P, Stein RA, DeFronzo RA, Shulman RG:
Quantitation of muscle glycogen synthesis in normal subjects and subjects with
non-insulin-dependent diabetes by 13C nuclear magnetic resonance
spectroscopy. N Engl J Med 322:223
-228, 1990[Abstract]
-
Oscai LB, Essig DA, Palmer WK: Lipase regulation of muscle
triglyceride hydrolysis. J Appl Physiol69
: 1571-1577,1990[Abstract/Free Full Text]
-
Falholt K, Jensen I, Lindkaer Jensen S, Mortensen H, Volund A,
Heding LG, Petersen PN, Falholt W: Carbohydrate and lipid metabolism of
skeletal muscle in type 2 diabetic patients. Diabet
Med 5: 27-31,1988[Medline]
-
Man ZW, Hirashima T, Mori S, Kawano K: Decrease in triglyceride
accumulation in tissues by restricted diet and improvement of diabetes in
Otsuka Long-Evans Tokushima fatty rats, a non-insulin-dependent diabetes
model. Metabolism 49:108
-114, 2000[Medline]
-
Storlien LH, Jenkins AB, Chisholm DJ, Pascoe WS, Khouri S, Kraegen
EW: Influence of dietary fat composition on development of insulin resistance
in rats: relationship to muscle triglyceride and omega-3 fatty acids in muscle
phospholipid. Diabetes 40:280
-289, 1991[Abstract]
-
Pan DA, Lillioja S, Kriketos AD, Milner MR, Baur LA, Bogardus C,
Jenkins AB, Storlien LH: Skeletal muscle triglyceride levels are inversely
related to insulin action. Diabetes46
: 983-988,1997[Abstract]
-
Lawrence JC Jr, Piper RC, Robinson LJ, James DE: GLUT4 facilitates
insulin stimulation and cAMP-mediated inhibition of glucose transport.
Proc Natl Acad Sci U S A 89:3493
-3497, 1992[Abstract]
-
Ren JM, Marshall BA, Mueckler MM, McCaleb M, Amatruda JM, Shulman
GI: Overexpression of Glut4 protein in muscle increases basal and
insulin-stimulated whole body glucose disposal in conscious mice. J
Clin Invest 95:429
-432, 1995[Medline]
-
Ren JM, Marshall BA, Gulve EA, Gao J, Johnson DW, Holloszy JO,
Mueckler M: Evidence from transgenic mice that glucose transport is
rate-limiting for glycogen deposition and glycolysis in skeletal muscle.
J Biol Chem 268:16113
-16115, 1993[Abstract/Free Full Text]
-
Chang PY, Jensen J, Printz RL, Granner DK, Ivy JL, Moller DE:
Overexpression of hexokinase II in transgenic mice: evidence that increased
phosphorylation augments muscle glucose uptake. J Biol
Chem 271:14834
-14839, 1996[Abstract/Free Full Text]
-
Manchester J, Skurat AV, Roach P, Hauschka SD, Lawrence JC Jr:
Increased glycogen accumulation in transgenic mice overexpressing glycogen
synthase in skeletal muscle. Proc Natl Acad Sci U S A93
: 10707-10711,1996[Abstract/Free Full Text]
-
Cheung JY, Conover C, Regen DM, Whitfield CF, Morgan HE: Effect of
insulin on kinetics of sugar transport in heart muscle. Am J
Physiol 234:E70
-E78, 1978[Abstract/Free Full Text]
-
Manchester J, Kong X, Nerbonne J, Lowry OH, Lawrence JC Jr: Glucose
transport and phosphorylation in single cardiac myocytes: rate-limiting steps
in glucose metabolism. Am J Physiol266
: E326-E333,1994[Abstract/Free Full Text]
-
Kubo K, Foley JE: Rate-limiting steps for insulin-mediated glucose
uptake into perfused rat hindlimb. Am J Physiol250
: E100-E102,1986[Abstract/Free Full Text]
-
Yki-Jarvinen H, Young AA, Lamkin C, Foley JE: Kinetics of glucose
disposal in whole body and across the forearm in man. J Clin
Invest 79:1713
-1719, 1987[Medline]
-
Katz A, Sahlin K, Broberg S: Regulation of glucose utilization in
human skeletal muscle during moderate dynamic exercise. Am J
Physiol 260:E411
-E415, 1991[Abstract/Free Full Text]
-
Schaffer JE, Lodish HF: Expression cloning and characterization of
a novel adipocyte long chain fatty acid transport protein.
Cell 79:427
-436, 1994[Medline]
-
Brindley DN: Metabolism of triacylglycerols. In
Biochemistry of Lipids, Lipoproteins, and Membranes: New
Comprehensive Biochemistry. Vol. 20. Vance
DE, Vance JE, Eds. Amsterdam, Elsevier Science, 1991, p.171
-203
-
West J, Tompkins CK, Balantac N, Nudelman ED, Meengs B, White T,
Bursten S, Coleman J, Kumar A, Singer JW, Leung DW: Cloning and expression of
two human lysophosphatidic acid acyltransferase cDNAs that enhance
cytokine-induced signaling responses in cells. DNA Cell
Biol 16: 691-701,1997[Medline]
-
Student AK, Hsu RY, Lane MD: Induction of fatty acid synthetase
synthesis in differentiating 3T3-L1 preadipocytes. J Biol
Chem 255:4745
-4750, 1980[Abstract/Free Full Text]
-
Coleman J: Characterization of Escherichia coli cells
deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity.
J Biol Chem 265:17215
-17221, 1990[Abstract/Free Full Text]
-
Hamilton S, Hamilton RJ, Sewell PA: Extraction of lipids and
derivative formation. In Lipid Analysis: A Practical
Approach. Hamilton RJ, Hamilton S, Eds. Oxford, NY, IRL Press at
Oxford University Press, 1992, p.13
-64
-
Hess SL, Suchin CR, Saltiel AR: The specific protein phosphatase
inhibitor okadaic acid differentially modulates insulin action. J
Cell Biochem 45:374
-380, 1991[Medline]
-
Harlow E, Lane D: Antibodies: A Laboratory
Manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory,1988
, p. 451
-
Kahn BB: Lilly Lecture 1995: Glucose transport: pivotal step in
insulin action. Diabetes 45:1644
-1654, 1996[Abstract]
-
Pendergrass M, Koval J, Vogt C, Yki-Jarvinen H, Iozzo P, Pipek R,
Ardehali H, Printz R, Granner D, DeFronzo RA, Mandarino LJ: Insulin-induced
hexokinase II expression is reduced in obesity and NIDDM.
Diabetes 47:387
-394, 1998[Abstract]
-
Lawrence JC Jr, Skurat AV, Roach PJ, Azpiazu I, Manchester J:
Glycogen synthase: activation by insulin and effect of transgenic
overexpression in skeletal muscle. Biochem Soc Trans25
: 14-19,1997[Medline]
-
Melki SA, Abumrad NA: Expression of the adipocyte fatty
acid-binding protein in streptozotocin-diabetes: effects of insulin deficiency
and supplementation. J Lipid Res34
: 1527-1534,1993[Abstract]
-
Weiner FR, Smith PJ, Wertheimer S, Rubin CS: Regulation of gene
expression by insulin and tumor necrosis factor alpha in 3T3-L1 cells:
modulation of the transcription of genes encoding acyl-CoA synthetase and
stearoyl-CoA desaturase-1. J Biol Chem266
: 23525-23528,1991[Abstract/Free Full Text]
-
Shin DH, Paulauskis JD, Moustaid N, Sul HS: Transcriptional
regulation of p90 with sequence homology to Escherichia coli
glycerol-3-phosphate acyltransferase. J Biol Chem266
: 23834-23839,1991[Abstract/Free Full Text]
-
Jerkins AA, Liu WR, Lee S, Sul HS: Characterization of the murine
mitochondrial glycerol-3-phosphate acyltransferase promoter. J Biol
Chem 270:1416
-1421, 1995[Abstract/Free Full Text]
-
Shapiro B, Chowers I, Rose G: Fatty acid uptake and esterification
in adipose tissue. Biochim Biophys Acta23
: 115-120,1957
-
Cahill GFJ, Leboeuf B, Renold AE: Factors concerned with the
regulation of fatty acid metabolism by adipose tissue. Am J Clin
Nutr 8: 733-739,1960
-
Kruszynska YT: Normal metabolism: the physiology of fuel
homoeostasis. In Textbook of Diabetes. Vol.1
, 2nd ed. Pickup JC, Williams G, Eds. Oxford,
Blackwell Science, 1997,11.1
-11.37
-
Hamilton JA: Fatty acid transport: difficult or easy? J
Lipid Res 39:467
-481, 1998[Abstract/Free Full Text]
-
Civelek VN, Hamilton JA, Tornheim K, Kelly KL, Corkey BE:
Intracellular pH in adipocytes: effects of free fatty acid diffusion across
the plasma membrane, lipolytic agonists, and insulin. Proc Natl
Acad Sci U S A 93:10139
-10144, 1996[Abstract/Free Full Text]