Increased glucose uptake promotes oxidative stress and PKC-
activation in adipocytes of obese, insulin-resistant mice
Ilana Talior,1
Merav Yarkoni,1
Nava Bashan,2 and
Hagit Eldar-Finkelman1
1Department of Human Genetics and Molecular
Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978; and
2Department of Clinical Biochemistry, Faculty of
Health, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
Submitted 31 January 2003
; accepted in final form 20 April 2003
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ABSTRACT
|
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Increased oxidative stress is believed to be one of the mechanisms
responsible for hyperglycemia-induced tissue damage and diabetic
complications. In these studies, we undertook to characterize glucose uptake
and oxidative stress in adipocytes of type 2 diabetic animals and to determine
whether these promote the activation of PKC-
. The adipocytes used were
isolated either from C57Bl/6J mice that were raised on a high-fat diet (HF)
and developed obesity and insulin resistance or from control animals. Basal
glucose uptake significantly increased (8-fold) in HF adipocytes, and this was
accompanied with upregulation of GLUT1 expression levels. Insulin-induced
glucose uptake was inhibited in HF adipocytes and GLUT4 content reduced by 20%
in these adipocytes. Reactive oxygen species (ROS) increased twofold in HF
adipocytes compared with control adipocytes and were largely reduced with
decreased glucose concentrations. At zero glucose, ROS levels were reduced to
the normal levels seen in control adipocytes. The activity of PKC-
increased twofold in HF adipocytes compared with control adipocytes and was
further activated by H2O2. Moreover, PKC-
activity was inhibited in HF adipocytes either by glucose deprivation or by
treatment with the antioxidant N-acetyl-L-cysteine. In
summary, we propose that increased glucose intake in HF adipocytes increases
oxidative stress, which in turn promotes the activation of PKC-
. These
consequential events may be responsible, at least in part, for development of
HF diet-induced insulin resistance in the fat tissue.
oxidative stress; protein kinase C-
; insulin resistance; glucose metabolism; C57Bl/6J mice
REACTIVE OXYGEN SPECIES (ROS) and the oxidative damage that they
cause are thought to play a key role in the pathogenesis of diabetes and its
complications (5,
18). It has been proposed that
hyperglycemia contributes to the generation in various tissues of free
radicals that, in turn, can lead to multiple complications and, at least in
part, to the induction of muscular and adipocyte insulin resistance
(10,
15,
39). It has been demonstrated
that chronic exposure of cells (such as endothelial cells, mesangial cells, or
smooth muscle cells) to high glucose increases intercellular ROS
(11,
22,
45,
47,
56). On the other hand,
oxidative stress could impair insulin action. Treatment of cells such as L6
myotubes or 3T3-L1 adipocytes with hydrogen peroxide
(H2O2) inhibited insulin-induced glucose uptake and
altered the translocation of GLUT4 to the plasma membrane
(6,
32,
52). The mechanism by which
oxidative stress causes insulin resistance is not fully clear. Part of the
explanation may stem from the fact that ROS affects various components of the
insulin-signaling cascade, as well as the gene regulation of the glucose
transporters GLUT1 and GLUT4
(5,
9,
32,
52,
63). In addition, ROS has been
shown to activate Syk (49,
55), Lck
(23), and the stress-activated
protein kinases p38 (6) and
c-Jun NH2-terminal kinases
(36) and protein kinase C
(PKC) (30) in various cell
types.
The novel PKC-
is one of the multifunctional isoenzymes of protein
kinase C, a family of serine/threonine kinases involved in intracellular
signals regulating growth and metabolism, differentiation, and apoptosis.
PKC-
itself was implicated in multiple cellular processes, some of
which were related to diabetes. In cultured muscle cells, PKC-
was
shown to be a major signal in insulin-induced glucose transport
(8) and to interact with the
insulin receptor in response to insulin
(51). Furthermore,
overexpression of PKC-
in skeletal muscle cultures abrogated the
inhibitory effect of tumor necrosis factor-
(TNF-
) on the
insulin-induced insulin receptor autophosphorylation
(51). Overexpression of
PKC-
in 3T3-L1 adipocytes enhanced basal glucose uptake
(61) but did not promote GLUT4
translocation when expressed in primary rat adipocytes
(2). In mesangial cells,
hyperglycemia increased the membrane- and particulate-associated PKC-
(28), and in vascular cells of
diabetic rats its cellular localization differed from that of normal animals
(25). Recently, PKC-
was implicated in oxidative stress and shown to be activated by
H2O2. This activation was accompanied by enhanced
phosphorylation of serine and tyrosine residues
(30,
31).
Oxidative stress is increased in cultured primary adipocytes exposed to
high glucose (37) and was
shown both to inhibit insulin sensitivity and to alter the expression levels
of glucose transporters in 3T3-L1 adipocytes
(52). To the best of our
knowledge, however, increased ROS production in adipocytes isolated from
diabetic animals has not been documented. In the present study, we undertook
to characterize glucose metabolism in adipocytes isolated from obese,
insulin-resistant C57Bl/6J mice, an inbred mouse strain susceptible to
diet-induced obesity and diabetes
(60), and to examine whether
this is linked to increased oxidative stress and changes in PKC-
activity. The present studies indicate that glucose uptake is significantly
increased in high-fat (HF) adipocytes and this, in turn, promotes oxidative
stress and activation of PKC-
.
 |
MATERIALS AND METHODS
|
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Materials. Insulin was a gift from Novo Nordisk (Bagsværd,
Denmark). The radioactive materials were purchased from Amersham Pharmacia
Biotech (Piscataway, NJ). GLUT1 and GLUT4 antibodies were from Chemicon
International (Temecula, CA), PKC-
and PKC-
antibodies were from
Santa Cruz Biotechnology (Santa Cruz, CA), and the phospho-PKC-
(Ser643) and PKB antibody were from Cell Signaling (Beverly, MA).
Insulin receptor and phospho-PKC-
(Ser657) antibodies were
from Upstate Biotechnology (Lake Placid, NY). All other reagents were
purchased from Sigma (Rehovot, Israel).
Animal and diets. Four-week-old mice were randomly assigned to
receive either standard laboratory food or a high-fat diet containing 35% lard
(Bioserve, Frenchtown, NJ), in which 55% of the calories came from fat. The
animals were housed in individual cages with free access to water in a
temperature-controlled facility with a 12:12-h light-dark cycle; the animals
were weighed periodically. Animals were used for testing after 16 wk on their
designated diets. Animals were starved for 6 h before experiments and then
killed. Blood samples were taken immediately from the aorta. Plasma glucose
was measured by the Sugar Accutrend Sensor (Roche, Mannheim, Germany), and
plasma insulin was assayed with a radioimmunoassay kit (INSIK-5, DiaSorin,
Saluggia, Italy). Animal care followed the institutional animal care and use
committee guidelines.
Preparation of adipocytes. To obtain an equal number of cells, the
protocol required pooled cells harvested from three to four lean mice for each
obese mouse. Adipocytes were isolated from the epididymal fat pad by digestion
with 0.4 mg/ml collagenase (Worthington Biochemical, Lakewood, NJ), as
described previously (33).
Digested fat pads were passed through nylon mesh, and the cells were washed
three times with Krebs-bicarbonate buffer (pH 7.4) that contained 1% bovine
serum albumin (fraction V; Boehringer Mannheim, Mannheim, Germany), 10 mM
HEPES (pH 7.3), 5 mM glucose, and 200 nM adenosine. Aliquots of cells were
used to determine the cell concentrations, as described
(14), and to determine the
amount of genomic DNA (Wizard Genomic; Promega, Madison, WI).
Histology and microscopy. Epididymal fat tissues were fixed in 4%
buffered formalin and embedded in paraffin. The paraffin-embedded tissue was
sectioned (4 µm) and stained with hematoxylin and eosin (Bio Optica, Milan,
Italy). Micro-graphs were taken at x100 magnification.
Glucose transport. Cells (1 x 106) in 1- to
1.5-ml aliquots were divided in plastic vials and incubated with shaking at
37°C. For glucose transport measurements, cells were incubated with
insulin at the indicated concentrations for 1 h, followed by the addition of
2-deoxy-[3H]glucose (0.5 µCi/vial) for 30 min. The assay was
terminated by the centrifugation of cells through dinonylphthalate (Irvine,
CA), and 3H was quantitated with a liquid scintillation analyzer
(Packard). Nonspecific uptake of 2-deoxy-[3H]glucose was determined
by the addition of cytochalasin B (50 µM) 30 min before the addition of the
radioactive material.
Total membrane and cell lysate preparations. Proteins were
extracted from the adipocytes or from fat tissue with buffer G (25
µg/ml each of 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM
-glycerophosphate, 50 mM NaF, 5% glycerol, 1% Triton X-100, and
leupeptin, aprotinin, and pepstatin A). After centrifugation, supernatants
were collected and subjected to gel electrophoresis followed by immunoblot
analysis with the indicated antibodies according to the manufacturer's
procedures. Total membranes were prepared from adipocytes of control and
diabetic animals (5 x 106 cells) as previously described
(46), with some modifications.
In brief, adipocytes were homogenized in buffer C (50 mM HEPES, pH
7.3, 10 mM NaF, 10 mM KCl, and 0.1 mM Na3VO4 and 25
µg/ml each of protease inhibitors leupeptin, aprotinin, and pepstatin A) by
30 strokes and centrifuged at 1,000 g at 4°C to remove nuclear
pellet. The resulting supernatants were centrifuged at high speed (100,000
g) at 4°C to precipitate membranes. Membranes were resuspended
with buffer C (100 µl), and equal aliquots of membranes (30 µl)
were subjected to gel electrophoresis followed by immunoblot analysis.
Measurement of intracellular ROS generation. The determination of
ROS was based on the oxidation of 2',7'-dichlorodihydrofluorescin
(DCHF) by peroxide, as previously described, with some modifications
(59). In brief, adipocytes
were incubated either with various concentrations of glucose (05 mM),
or with N-acetyl-L-cysteine (NAC; 0.1, 0.2, or 0.4 mM) for
1 h and then incubated with DCHF (30 µM) for an additional 40 min.
Incubation with H2O2, a ROS donor, served as a positive
control, and known concentrations of DCHF incubated with 20 mM NaOH were used
as standards. Cells were washed, and the fluorescence of
2',7'-dichlorofluorescein (DCF) was measured in triplicate samples
in a multiplate fluorometer (Fl-600; excitation at 488 nm, emission at 530
nm).
PKC-
activity. PKC-
activity was assayed as
previously described (30). In
brief, epididymal fat tissues were removed from both the control and the HF
mice and immediately homogenized with buffer H (25 µg/ml each of
20 mM Tris, pH 7.5, 50 mM
-glycerophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM
DTT, 10 mM NaF, 5% glycerol, 1% Triton X-100, and leupeptin, aprotinin, and
pepstatin A). A similar procedure was performed in isolated adipocytes, except
that cells were not homogenized. The lysates were centrifuged, and equal
protein aliquots were used for PKC-
assays. The enzyme was
immunoprecipitated for 2 h with anti-PKC-
antibody bound to protein
Sepharose. The immunocomplexes were washed extensively, and the kinase
reaction was performed at 30°C in 20 mM Tris · HCl, pH 7.3, with 10
mM MgCl2, 100 µM [
-32P]ATP, 40 µM
phosphatidylserine, and 3 µg histone H1 (per reaction). Reactions were
terminated after 20 min by the addition of SDS-PAGE load buffer and were
analyzed in 15% acrylamide gels by autoradiography. Autoradiography was
quantitated by densitometry.
Expression of results. Results are expressed as means ± SE
for the number of cell preparations or animals, as indicated. Statistical
analysis was performed by Origin Professional 6.0, using one-way ANOVA to
compare the control and HF results. A difference was considered to be
statistically significant when P < 0.05.
 |
RESULTS
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Characterization of HF mice and HF adipocytes.
Table 1 characterizes C57BL/6J
mice, which were fed the HF diet, and their respective controls, which were
fed standard laboratory food. After 16 wk on their designated diets, the
animals on the HF diet developed hyperglycemia and hyperinsulinemia. The HF
mice increased their weight by 3040% compared with control animals.
Notably, there was about a fourfold increase in the number of adipocytes in
each epididymal fat pad of HF mice compared with their respective controls.
Determination of genomic DNA further confirmed this result and indicated that
DNA content increased about fourfold in adipocytes isolated from HF fat pads
compared with that of control (0.2 vs. 0.05 µg/fat pad of HF or control,
respectively). Determination of the protein content of the fat tissues
(Table 1) indicated that
protein content also increased approximately fourfold in the HF tissue
compared with control. Hence, protein content per cell did not significantly
change in HF adipocytes compared with control adipocytes (calculated value 4
ng/cell), suggesting that the increase in the HF adipocytes size
[
2.5-fold (cell diameter); Fig.
1] results mainly from accumulation of lipids.

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Fig. 1. Fat tissue morphology of high-fat diet-fed and control mice. Representative
adipose tissue paraffin sections of the control (C) or obese diabetic (HF)
mice stained with hematoxylin and eosin. Micro-graphs were taken at initial
magnification of x100.
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Increased glucose uptake in HF adipocytes is accompanied by GLUT1
upregulation. Basal glucose uptake was strikingly higher in adipocytes
from the HF mice compared with the controls (8.6-fold;
Fig. 2A) and was blocked with
cytochalasin B, indicating that glucose uptake is specific (data not shown).
Insulin-stimulated glucose uptake is approximately threefold higher in control
adipocytes, but insulin had no stimulatory effect in adipocytes from diabetic
animals (Fig. 2A).
This is consistent with studies performed in adipocytes derived from type 2
diabetic animals, showing a significant increase in basal glucose uptake and
inhibition in insulin-stimulated glucose uptake
(13,
50). Analysis of GLUT1
expression levels in total membranes prepared from an equal number of
adipocytes indicated that GLUT1 is consistently higher in HF adipocytes
(Fig. 2B). The
elevation in GLUT1 in HF adipocytes seems to be specific, because the amount
of another membrane protein, the insulin receptor, was not elevated but rather
was reduced in HF adipocytes compared with controls
(Fig. 2C). The
expression level of GLUT4, which is known to be located in intracellular
membranes, was reduced by 20 ± 4%, whereas no change in the cytosolic
PKB was observed in either control or HF adipocytes
(Fig. 2, D and
E). Notably, alterations in GLUT1 and GLUT4 content were
also observed in adipocytes of Zucker diabetic rats
(46). All together, our
results suggest that the increase in basal glucose uptake in HF adipocytes
stems from specific upregulation of GLUT1 and is not due to a general
phenomenon, namely increased protein expression levels, because other proteins
were not elevated.

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Fig. 2. Glucose uptake, GLUT1, GLUT4, insulin receptor (IR), and protein kinase B
content in adipocytes from HF and control animals. A:
2-deoxy-[3H]glucose uptake in absence and presence of various
insulin concentrations. Results are presented as 2-deoxyglucose incorporation
into 106 cells in 1 min and are means ± SE of 5 independent
experiments. B: GLUT1 protein content was measured in total membrane
prepared from 5 x 106 adipocytes by Western blot analysis as
described in MATERIALS AND METHODS. Shown are representative blot
(top) and densitometry analysis (bottom) of independent
experiments in which GLUT1 level in controls was assigned a value of 1.
*P < 0.05 C vs. HF. C: IR protein content in
total membrane preparation. D: GLUT4 protein content in total cell
lysates was determined by Western blot analysis as described in MATERIALS
AND METHODS. E: PKB protein content in total cell lysate was
determined by Western blot analysis as described in MATERIALS AND
METHODS. In C, D, and E, representative blots
performed 4 times are shown. Cell lysate and membranes were prepared as
described in MATERIALS AND METHODS, and equal amounts of membranes
(corresponding to 1.6 x 106 cells) or cell lysates
(corresponding to 1.50 x 105 cells) were loaded onto
gels.
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Intracellular ROS is increased in HF adipocytes. We next examined
whether oxidative stress is increased in HF adipocytes and whether this may be
related to the enhanced glucose uptake of these adipocytes. Intracellular ROS
measurements presented in Fig.
3A indicate that ROS levels increased twofold in HF
adipocytes compared with the control cells. Incubation of cells with a
glucose-free medium resulted in a 50% decrease in ROS levels in the HF
adipocytes (Fig. 3A).
Figure 3B demonstrates
the production of ROS in HF adipocytes as a function of medium glucose
concentrations. As can be seen, elevation in ROS production correlates with
elevation in medium glucose concentration, suggesting that elevation in
glucose uptake in HF adipocytes is a major contributor to ROS production.

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Fig. 3. Reactive oxygen species (ROS) levels in HF and control adipocytes. DCF,
2',7'-dichlorofluorescein. A: ROS levels were determined
in control cells after 1-h incubation in glucose-containing medium and in HF
adipocytes incubated for 1 h in presence or absence of glucose, as described
in MATERIALS AND METHODS. *P < 0.05 C vs. HF
or HF incubated in absence vs. HF presence of glucose. Results represent means
± SE of 5 independent experiments. B: same as A,
except that HF adipocytes were incubated with various concentrations of
glucose (1 h) as indicated. Results represent means ± SE of 3
independent experiments.
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PKC-
activity is increased in HF adipocytes.
Previous studies demonstrated that PKC-
is activated in various cell
types by oxidative stress (30,
31). Thus we evaluated both
PKC-
activity (using histone H1 as substrate) and the abundance of
PKC-
and its phosphorylation state in adipocytes derived from both HF
and control mice (Fig. 4). As
can be evaluated (Fig.
4A), PKC-
activity was higher in HF adipocytes
compared with controls. Immunoblot analysis with the anti-phospho-PKC-
antibody, which recognizes the phosphorylated Ser643, a site known
to be essential for the activation of PKC-
when phosphorylated
(35) and is phosphorylated in
response to H2O2
(31), indicated that
phosphorylation of Ser643 incresaed in HF adipocytes compared with
controls with no change in the expression of PKC-
(Fig. 4, B and
C). The phosphorylation of the conventional PKC-
isoform on Ser657, a site known to be crucial for the enzyme
activity when phosphorylated
(7), and its expression levels
were similar in HF and control adipocytes
(Fig. 4D).
PKC-
activity is activated by
H2O2 and inhibited
by depletion of glucose and NAC. To further investigate the link between
ROS and PKC-
, the effect of H2O2 on PKC-
in HF adipocytes was studied. As shown in
Fig. 5A, exposure to
H2O2 (200 µM or 2 mM) activated PKC-
,
suggesting that the increase in ROS production can contribute to the
activation of PKC-
in HF adipocytes. Furthermore, when cells were
incubated with glucose-free medium (1 h), PKC-
activity was greatly
inhibited (Fig. 5B).
To further asses the role of ROS in activating PKC-
, we used NAC, a
known free-radical scavenger previously shown to deplete intracellular ROS
levels in adipocytes (37).
Treatment of HF adipocytes with increasing NAC concentrations reduced ROS
production (Fig. 5C)
and basal PKC-
activity (Fig.
5D). Taken together, these results suggest that increased
oxidative stress in HF adipocytes is dependent on glucose efflux and promotes
the activation of PKC-
.
 |
DISCUSSION
|
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Numerous reports have shown that oxidative stress is increased in animal
models of diabetes and in patients with diabetes
(5,
18). In this study, we used an
ex vivo system of isolated adipocytes from diabetic mice (HF adipocytes) to
investigate whether oxidative stress is increased in these adipocytes and
whether this may be related to hyperglycemia and/or activation of PKC. We
showed that glucose uptake and ROS levels increased significantly in HF
adipocytes compared with control adipocytes. Subsequently, ROS levels were
reduced by decreased medium glucose concentrations in HF adipocytes, and in
the absence of glucose its levels were reduced twofold, reaching the
"normal" ROS levels observed in the control adipocytes. In
addition, PKC-
activity was higher in HF adipocytes compared with
controls and could be further activated by H2O2.
Moreover, the enzyme activity was inhibited by either the antioxidant NAC or
the depletion of glucose. Taken together, these results enabled us to propose
that the increase in glucose efflux into HF adipocytes may increase
intracellular ROS production that, in turn, promoted the activation of
PKC-
. These intracellular events could represent, at least in part, one
of the mechanisms responsible for induction of insulin resistance in the fat
tissue. It should be noted, however, that our studies do not eliminate the
role of additional in vivo factors such as hyperinsulinemia, hyperlipidemia,
and adipocytokines that could contribute to the increased oxidative stress
during the HF diet feeding period, as these factors were shown to elevate ROS
levels in various cell systems
(1,
5,
17,
20,
29,
42,
44,
64).
GLUT1 levels were significantly increased in HF adipocytes
(Fig. 2B). Previous
studies demonstrated that GLUT1 expression levels might be affected by stress
conditions such as hyperglycemia, hyperinsulinemia, or TNF-
(24,
34,
40). Although we do not know
the exact mechanisms leading to increased GLUT1 expression in HF adipocytes,
we suggest that the elevation in GLUT1 was responsible for the increased
glucose uptake of HF adipocytes, which in turn led to the elevation of ROS. On
the other hand, ROS production could further lead to increased GLUT1
transcription rates. The latter supposition is based on previous studies that
indicated that induction of oxidative stress in 3T3-L1 adipocytes or L6 muscle
cells resulted in elevation of GLUT1
(32,
52). Thus it is suggested that
a vicious cycle may be operating between GLUT1 content and increased oxidative
stress in HF adipocytes.
The generation of ROS as a byproduct of the mitochondrial electron
transport chain has long been attributed to the high rates of glucose
metabolism (4,
16,
54,
63). Nevertheless, additional
mechanisms may be operating to produce ROS under hyperglycemic conditions,
including the formation of advanced glycation end products
(5,
42) or activation of oxidases
such as NADPH oxidase (26,
41,
62). We do not know at this
point the precise source of the production of ROS in HF adipocytes. Further
studies should elucidate this problem.
Our studies indicated that insulin receptor levels significantly decreased
in HF adipocytes (Fig.
2C). This phenomenon was reported in various tissues and
cells in conditions associated with insulin resistance
(21,
38,
65). Hyperinsulinemia has been
suggested to be a prominent factor responsible for this phenomenon
(27,
48), thereby indicating that
downregulation of insulin receptors is a secondary effect in insulin
resistance. Nevertheless, it is still possible that reduced levels of insulin
receptors in HF adipocytes could limit the sensitivity of the cells to
insulin. On the other hand, it should be noted that the remaining insulin
receptors (
50%) could still transmit intracellular insulin signaling, as
the receptors are most likely not defected
(38).
The novel PKC isoform PKC-
was shown to be activated by oxidative
stress in various cell systems. Initially, it was shown that
H2O2 led to activation in COS7 cells of PKC-
,
which was constitutively active and independent of the lipid coactivator,
diacylglycerol (30,
31). Additionally, oxidative
stress was shown to stimulate PKC-
and c-Abl tyrosine kinase
association, facilitating the activation of c-Abl by PKC-
(58). In vascular smooth
muscle cells, H2O2 activation of PKC-
was
required for the activation of platelet-derived growth factor receptor
(53). PKC-
was also
shown to mediate oxidative stress-induced activation of several signaling
components, including the nonreceptor tyrosine kinases JAK2 and PYK2
(19), as well as the
serine/threonine protein kinase MAPK
(12). Thus PKC-
may
play a central role in oxidative stress-induced cellular processes important
in the development of insulin resistance in HF adipocytes. Further studies are
needed to examine the downstream targets activated by PKC-
in these
cells.
PKC was initially shown to play a role in glucose transport in primary rat
adipocytes (43,
57). Overexpression of
PKC-
in 3T3-L1 adipocytes enhanced both basal and insulin-induced
glucose transport (61).
Another study reported that PKC-
, PKC-
, PKC-
, and
PKC-
were not involved in insulin-induced GLUT4 translocation
(3). Thus it may be concluded
that elevated PKC-
activity could contribute to increased basal glucose
uptake in HF adipocytes; on the other hand, and in agreement with previous
findings (3), it has no effect
on insulin-induced GLUT4 translocation in these adipocytes.
In summary, we suggest that the consequential events of glucose uptake, ROS
production, and activation of PKC-
may represent one of the mechanisms
responsible for the development of insulin resistance in HF adipocytes in
response to HF diet feeding.
 |
DISCLOSURE
|
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This work was supported by the Israeli Diabetes Foundation and by the
Annual Award of the Hendrik and Irene Gutwirth Research Prize in Diabetes
Mellitus, which was awarded to H. Eldar-Finkelman.
 |
ACKNOWLEDGMENTS
|
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We thank Prof. Zvi Nevo from the Department of Clinical Biochemistry for
conducting the histological studies.
 |
FOOTNOTES
|
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Address for reprint requests and other correspondence: H. Eldar-Finkelman,
Dept. of Human Genetics and Molecular Medicine, Sackler School of Medicine,
Tel Aviv University, Tel Aviv 69978, Israel (E-mail:
heldar{at}post.tau.ac.il).
Submitted 31 January 2003
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.
 |
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