| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461; USA; and
Department of Animal Sciences, Purdue University, West Lafayette, Indiana 47907, USA
1Correspondence: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA. E-mail: charron{at}aecom.yu.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: adipocyte glucose transport • NIDDM • hyperglycemia • PI3 kinase
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2
3)
. Diseases such as non-insulin-dependent diabetes
mellitus (NIDDM) and obesity are characterized by insulin resistance
(3
, 4)
. Insulin signaling pathways involving the
activation of PI3-kinase are responsible for the metabolic regulation
of carbohydrate, lipid, and protein utilization (5)
. These
pathways are logical targets for defects associated with insulin
resistance and diabetes. However, there is a paucity of data concerning
the progression of insulin signaling defects with the onset of insulin
resistance and diabetes (2)
. This is especially true for
the signaling events in adipocytes.
The GLUT4 heterozygous knockout mouse, GLUT4(+/-), is a novel mouse
model for studying the development of insulin resistance and diabetes
(6)
. In an outbred genetic background, male GLUT4(+/-)
mice display the full spectrum of phenotypes leading to insulin
resistance and NIDDM (6)
. At all ages there are
GLUT4(+/-) mice with normal fed glycemia and insulinemia (N/N), normal
fed glycemia and hyperinsulinemia (N/H), and fed hyperglycemia with
hyperinsulinemia (H/H). Whereas 53% of males were able to maintain
normal glucose and insulin levels at ages of around 2 to 4 months, more
than 85% of male GLUT4(+/-) mice develop fed hyperinsulinemia, and
60% of them develop fed hyperglycemia at 5 to 7 months of age
(6)
. The diabetic H/H GLUT4(+/-) mice have hypertension,
diabetic cardiomyopathy, and reduced insulin-stimulated skeletal muscle
glucose uptake but not dyslipidemia or obesity (6)
. In
addition, prediabetic N/H GLUT4(+/-) mice have severely reduced whole
body glucose turnover, glycolysis, glycogen synthesis, and insulin
action but normal hepatic glucose fluxes (7)
. It is the
successive development of fed hyperinsulinemia, followed by
hyperglycemia, that makes the male GLUT4(+/-) mice an exceptional
model for studying progressive changes in insulin signaling pathways
associated with diabetic pathogenesis. Furthermore, the absence of
obesity in these mice makes it possible to dissociate the effect of
obesity on the diabetic phenotype, which complicates analysis in
existing animal models of diabetes (8)
.
Insulin has diverse functions in different tissues and cell types
reflected by the complicated signaling cascade (5
, 9
, 10)
.
The binding of insulin to its receptor activates the tyrosine kinase
activity of the insulin receptor (IR), which leads to tyrosine
phosphorylation of IR itself and the IR substrates IRS-1, IRS-2, Src
homology collagen (Shc), and pp60/IRS-3 (2
, 9
, 11)
.
Tyrosine-phosphorylated substrates then recruit other proteins such as
the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) and Grb
protein via SH2 domains (9
, 10)
. It has been shown that
the activation of PI3 kinase leads to translocation of GLUT4-containing
vesicles and subsequently increased glucose uptake in skeletal muscle
and adipocytes (1
, 12)
.
Studies examining insulin signaling defects in skeletal muscle,
liver, and adipose tissue of insulin-resistant animal models and obese
or diabetic human subjects have shown reduced insulin-stimulated IR and
IRS-1 tyrosine phosphorylation and decreased insulin-stimulated,
IRS-1-associated PI3 kinase activity (13
14
15
16
17
18
19)
. Studies
with IRS-1-deficient mice suggested that insulin-stimulated,
IRS-1-associated PI3 kinase activity is responsible for
insulin-stimulated glucose uptake in skeletal muscle (20)
.
However, in adipose tissue, the role of insulin-stimulated,
IRS-1-associated PI3 kinase activity in determining insulin-stimulated
glucose uptake is not clear (21)
. Recently, Rondinone and
colleagues (22)
demonstrated that whereas IRS-1 protein
expression is reduced, IRS-2 becomes the main docking protein for PI3
kinase in adipocytes from NIDDM patients.
Reduced GLUT4 protein expression is a common characteristic of adipose
tissue from animal models of diabetes and insulin resistant
(23)
. However, down-regulation of GLUT4 protein expression
in adipocyte seems to be the consequence of the development of whole
body insulin resistance in obese insulin-resistant mice
(24)
. The contribution of decreased GLUT4 content
per se to adipocyte insulin resistance is unknown. To date,
no studies have described alternations in the insulin signaling cascade
in adipocytes during the development of diabetes.
GLUT4(+/-) mice present a unique opportunity to study the onset and
progression of insulin signaling defects concomitant with the
progression of whole body insulin resistance and diabetes
(6)
. As GLUT4 content is decreased first in adipocytes of
GLUT4(+/-) mice, when most of the animals have normal serum glucose
and insulin concentrations (6)
, it is possible to answer
the question of whether reduced GLUT4 protein content alone can cause
insulin resistance and insulin signaling defects. In the present study,
we describe the progression of defects in insulin signaling in
adipocytes from GLUT4(+/-) mice that accompany the pre- and overt
diabetic stages.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
.
The mice were housed in rooms with a regular day/night cycle and were
fed standard rodent chow and water ad libitum.
Blood glucose and plasma insulin determinations
Ad libitum fed mice were bled at ~1:30
A.M. for fed serum levels of glucose and insulin.
Whole blood was drawn from the orbital sinus and centrifuged
immediately. Serum was immediately frozen on dry ice and kept at
-70°C until further use. Serum glucose levels were measured by the
glucose oxidase method (Sigma, Inc., St. Louis, Mo.). Serum insulin
levels were measured by radioimmunoassay (Linco Research, Inc., St.
Louis, Mo.) using rat insulin standards.
Isolation of epididymal adipocyte
Mice were killed by cervical dislocation at around 11:00
A.M. and epididymal fat pads were dissected and weighed.
Adipocytes were isolated by collagenase digestion (Worthington, Inc.,
Freehold, N.J.; 1 mg/g fat pad) at 37°C as described previously
(26)
. Digested fat pads were washed and resuspended in
Krebs-Ringer-bicarbonate-HEPES (KRPH) buffer (pH 7.4) supplemented with
2.5% bovine serum albumin (BSA; Cohn fraction V, Intergen, Inc.,
Purchase, N.Y.) and 200 nM adenosine (26)
.
Immunoblotting
Isolated adipocytes were lysed in a buffer containing 50 mM
HEPES, 150 mM NaCl, 2.0 mM NaO3V, 20 mM NaF, 4 mM EDTA, 20% glycerol,
10% Nonidet P-40, 4 mM PMSF, 20 nM leupeptin, and 20 nM aprotinin.
Crude adipocyte lysates were used for immunoblot analysis. Proteins
were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), electrophoretically transferred to Hybond
ECL membrane (Amersham, Inc., Arlington, Ill.), and reacted with
specific antibodies. For GLUT4 protein detection, 30 µg of total
lysates were loaded and a polyclonal antibody against the carboxyl
terminus of GLUT4 was used (25)
. For detection of p85
protein, 40 µg of total lysates were loaded and an antibody against
the SH2 domain of p85 protein was used (a kind gift from Dr. J.
Backer). For detection of IR protein, 100 µg of total lysates were
loaded and an antibody against the carboxyl terminus of human IR ß
subunit was used (a kind gift from Dr. B. Cheatham). For detection of
IRS-1 protein, 2 mg of total lysates were used in immunoprecipitations
with an antibody specific against the PH domain of IRS-1 (a kind gift
from Dr. M. White) and then blotted using the same antibody.
Determination of adipose cell size and number
Resuspended adipose cells were aliquoted in two triplicate sets.
One set was fixed in 2% osmic acid, washed in distilled water, and
resuspended in 1 ml of counting solution that contained 55% (v/v)
glycerol, 1% (v/v) Triton X-100 and 3.85% NaCl. Triplicates (6 ml) of
the resuspended cells were counted using a hemocytometer under a
microscope. The other set of triplicates was used to determine cell
sizes. Cells were extracted using heptane and lipid content was
weighed. Adipose cell size was determined as microgram lipid/cell as
described previously (27
, 28)
.
Adipocyte glucose transport
Isolated adipocytes were incubated for 30 min in KRPH buffer
supplemented with 2.5% BSA at 37°C in the presence or absence of
various concentrations of porcine insulin (a kind gift from Dr. R.
Chance). [U-14C]glucose
(Amersham; 0.3 mM final concentration) uptake was performed as
described previously (26)
. Briefly,
[U-14C]glucose was added to the cell suspension
for 20 min and the reaction was terminated by spinning the cell
suspension through dinonylphthalate oil (ICN Biomedicals, Inc., Aurora,
Ohio). The upper phase, which contains adipose cells, was collected and
subjected to liquid scintillation counting. Under the assay conditions,
uptake of [14C]glucose has been shown to
directly reflect glucose transport (26)
.
Determination of PI3 kinase activity
Isolated adipocytes were divided into two groups: basal and
insulin stimulated. In the basal group, cells were immediately lysed in
lysis buffer. In the insulin-stimulated group, cells were incubated
with 100 nM insulin for 5 min prior to lysis. The lysate was
centrifuged at 2500 g for 5 min; 1 mg of solubilized lysate
was used for immunoprecipitations with 8 µl of anti-p85 or 10 µl of
anti-IRS-1 antibodies. Immunoprecipitation was carried out at
4C overnight with constant rotating. Protein
A-Sepharose beads were added and incubation was continued for 3 more
hours. Beads-associated PI3 kinase activity was assayed based on
phosphorylation of phosphatidylinositol in the presence of
[
-32P]ATP (Amersham) (14)
.
Reaction products were separated by thin layer chromatography (TLC),
exposed to X-ray film, and quantified by densitometry. Quantification
was also performed on representative plates by Cerenkov counting of
[32P] phosphate incorporated into PI(3)P. In
all cases, the fold difference was comparable to that measured by
densitometry. Activity of PI3 kinase was expressed as the percentage of
control activity in the basal state.
Immunoblot analysis of tyrosine-phosphorylated proteins on insulin
stimulation
One milligram of total adipose cell lysate protein from basal
and insulin-stimulated cells was immunoprecipitated with 10 ml of the
antiphosphotyrosine antibody PY20 (Transduction Labs, Inc., Lexington,
Ky.) for 4 h at 4°C. The immunocomplex was brought down by
protein A-Sepharose beads, loaded onto 8% SDS-PAGE, and immunoblotted
onto ECL membrane (Amersham). The filter was then reacted with the
antiphosphotyrosine antibody RC20 (Transduction Labs, Inc.) according
to the manufacturer’s instructions.
Statistical analysis
Data are presented as mean ± SE. Significance
between groups was determined by using the Student’s t
test. P<0.05 was accepted as statistically significant.
Pooled samples were treated as one for analysis.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Glucose between 200 and 220 mg/dl was categorized as normal glucose
while glycemia higher than 350 mg/dl was grouped as hyperglycemia.
Insulin concentrations lower than 10 ng/ml were considered normal
insulinemia whereas concentrations higher than 25 ng/ml were grouped as
hyperinsulinemia. The 3-month-old GLUT4(+/-) mice with normal fed
glucose and fed insulin levels were referred to as normal/normal (N/N).
The 3- to 4-month-old prediabetic GLUT4(+/-) mice with normal fed
glucose but high fed insulin were referred to as normal/high (N/H).
Since it was difficult to get enough animals with both hyperglycemia
and hyperinsulinemia at 3 to 4 months of age, 6- to 9-month-old
diabetic GLUT4(+/-) mice with both fed hyperglycemia and
hyperinsulinemia were used and referred to as high/high (H/H). Control
mice in each group were age and sex matched. All GLUT4(+/-) mice had
body and epididymal fat pad weights similar to their age-matched
controls (Table 2
). Epididymal fat cell size was similar in both normal N/N and
prediabetic N/H groups, but was 35% ± 0.02 larger in diabetic H/H
group compared to controls (Table 2)
.
|
|
Expression of GLUT4 protein in adipocytes of heterozygous
GLUT4(+/-) knockout mice
The expression of GLUT4 protein was assessed by Western blot
analysis using a specific antibody against GLUT4. GLUT4 protein content
in adipocytes was reduced by 50% ± 0.01 in all three groups
(Fig. 1
). In the diabetic H/H group, the reduction in GLUT4 was slightly
greater than the other two groups (Fig. 1C
). GLUT1 protein
content in the fat was measured in a previous study in the diabetic H/H
and control groups, and no significant difference in expression was
detected (6)
.
|
Glucose uptake in adipocytes of heterozygous GLUT4(+/-) knockout
mice
To delineate the effect of reduced GLUT4 expression and
whole body metabolic milieu on glucose uptake,
[U-14C]glucose accumulation in the absence or
the presence of 1 nM and 100 nM insulin concentrations was studied.
Basal glucose uptake was normal in adipocytes from all three groups of
GLUT4(+/-) mice. In the presence of 1 nM insulin, control cells for
both N/N and N/H adipocytes showed a significant increase in glucose
uptake. Adipocytes from N/N GLUT4(+/-) mice also showed a comparable
increase in glucose uptake at 1 nM of insulin. However, adipocytes from
N/H animals exhibited a 50% decrease in glucose uptake at 1 nM insulin
compared to controls. No significant increase in glucose uptake was
detected in the 6- to 9-month-old control and H/H groups in the
presence of 1 nM insulin (10.72±2.49 in the H/H adipocytes;
11.91±3.482 in the controls). Consequently, the insulin concentration
was increased to 5 nM for these groups. At this insulin concentration,
a significant increase in glucose uptake in the control adipocytes was
achieved, whereas an attenuated increase in glucose uptake in the
H/H adipocytes was seen (50% of control at the same insulin
concentration) (Fig. 2
).
|
The reduction of glucose uptake in N/H and H/H GLUT4(+/-) adipocytes under such concentrations of insulin stimulation suggested that insulin sensitivity was reduced in these cells. The maximal fold stimulation of glucose uptake by insulin was similar in all three groups of GLUT4(+/-) adipocytes (2.7-fold in N/N, 2.8-fold in N/H, and 2.6-fold in H/H). On the other hand, the maximal insulin-stimulated glucose uptake was 6.0- to 7.4-fold in controls. Thus, the relative fold decrease in maximal insulin-stimulated glucose uptake in all groups of GLUT4(+/-) adipocytes was 50%, which was the same as the reduction of GLUT4 protein content.
Insulin-stimulated protein tyrosine phosphorylation in adipocytes
of heterozygous GLUT4(+/-) knockout mice
The difference in insulin sensitivity among N/N, N/H, and H/H
GLUT4(+/-) adipocytes suggested modulation of insulin signaling in
these cells. Insulin-stimulated protein tyrosine phosphorylation
profiles were examined using isolated adipocytes from all three groups.
Tyrosine phosphorylation of three major bands that migrate at the
positions of the IR ß subunit, IRS-1, and IRS-3 (11
, 29)
were uniformly identified in controls. No changes in the intensity of
tyrosine phosphorylation of these three bands were detected in
adipocytes from N/N GLUT4(+/-) mice (Fig. 3A
). However, reductions (64%±5% of IR, 71%±4% of IRS-1,
and 35%±1% of IRS-3) were observed in adipocytes from N/H
GLUT4(+/-) mice (Fig. 3B
). Insulin-stimulated tyrosine
phosphorylation of IR ß subunit, IRS-1, and pp60/IRS-3 was further
reduced in the diabetic H/H GLUT4(+/-) adipocytes (Fig. 3C
). IRS-1 tyrosine phosphorylation was barely detectable on
immunoblot analysis of H/H adipocytes. Tyrosine phosphorylation of IR
ß subunit and pp60/IRS-3 in H/H GLUT4(+/-) adipocytes was reduced to
12% ± 1% and 23% ± 3% of control levels, respectively.
|
Expression of IR, IRS-1, and p85 in adipocytes of heterozygous
GLUT4(+/-) knockout mice
The total protein content of IR, IRS-1, and p85 was measured in
adipocytes from all three groups (Fig. 4
). No difference in the content of these proteins was noted in
adipocytes of either N/N or N/H groups compared to controls. However, a
42% ± 3% reduction of IR protein was noted in adipocytes from H/H
GLUT4(+/-) adipocytes (Fig. 4A
). Expression of p85 was
increased 80% ± 3% over controls in H/H GLUT4(+/-) adipocytes (Fig. 4B
).
|
Insulin-stimulated PI3 kinase activity in adipocytes of
heterozygous GLUT4(+/-) knockout mice
PI3 kinase is involved in mediating insulin-stimulated GLUT4
translocation and insulin-stimulated glucose uptake in adipocytes.
Insulin-stimulated, IRS-1-associated PI3 kinase activity was normal in
adipocytes from N/N GLUT4(+/-) mice compared to controls (Fig. 5A
). A modest decrease in IRS-1-associated PI3 kinase activity
of 20% ± 1% was noted in adipocytes from N/H GLUT4(+/-) mice (data
not shown). This was further decreased to 32% ± 1% of controls in
adipocytes from H/H GLUT4(+/-) mice (Fig. 5B
).
|
To understand the significance of increased p85 protein
expression in adipocytes of H/H GLUT4(+/-) mice, p85-associated PI3
kinase activity was studied. Basal levels of p85-associated PI3 kinase
activity in H/H GLUT4(+/-) adipocytes were 50% ± 3% higher than
controls (Fig. 6
). Similar p85-associated PI3 kinase activity was measured in H/H
GLUT4(+/-) compared to control adipocytes under maximal insulin
stimulation (Fig. 6)
.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 15
, 16
, 30)
.
Decreased insulin-stimulated IRS-1 phosphorylation and PI3 kinase
activity have been observed in adipocytes from human subjects with
NIDDM and gold thioglucose-induced obese hyperinsulinemic mice
(17
18
19)
. However, the earliest alterations in cellular
and molecular aspects of insulin action are poorly defined as very few
studies of prediabetic populations have been performed. In contrast,
individual or additive effects of hyperinsulinemia and hyperglycemia on
insulin signaling pathways have been suggested (4)
.
However, data are lacking defining the modulation of the insulin
signaling cascade in adipocytes after the onset of insulin resistance
and overt diabetes. On the other hand, decreased GLUT4 expression has
been uniformly observed in adipocytes from animal models of insulin
resistance and in obese and NIDDM patients (23)
.
Down-regulation of GLUT4 protein expression in adipocytes seems to be a
consequence of development of whole body insulin resistance in obese
insulin-resistant mice (24)
. However, the significance of
reduced GLUT4 content relative to adipocyte insulin sensitivity is not
well understood. It is unclear whether reduced GLUT4 protein or
impaired insulin signaling is the rate-limiting step in determining
insulin-stimulated glucose uptake in adipocytes.
Targeted disruption of the GLUT4 gene leads to different phenotypes in
homozygous and heterozygous knockout mice (6
, 25)
. The
existence of N/N, N/H, and H/H GLUT4(+/-) mice make them an ideal
system for studying the progressive development of diabetes
(6)
. In GLUT4(+/-) mice, the decrease in GLUT4 expression
was first detected in white adipose tissue of 2-month-old males,
preceding changes in skeletal muscle GLUT4 protein content
(6)
. GLUT4(+/-) mice at this age had normal fed glycemia
and insulinemia. Insulin signaling in adipocytes from these mice was
normal, as indicated by normal insulin-responsive protein tyrosine
phosphorylation and IRS-1-associated PI3 kinase activation (Fig. 3A
, Fig. 5A
). Studies of insulin-stimulated
protein phosphorylation in N/H and H/H GLUT4(+/-) adipocytes delineate
the progression of insulin signaling defects along with the development
of hyperinsulinemia and hyperglycemia. Decreased insulin-stimulated
protein tyrosine phosphorylation was observed in adipocytes once
GLUT4(+/-) mice developed hyperinsulinemia, as seen in N/H GLUT4(+/-)
adipocytes (Fig. 3B
). It was further decreased in H/H
GLUT4(+/-) adipocytes (Fig. 3C
). In addition, the
development of hyperglycemia was accompanied by decreased IR expression
in H/H GLUT4(+/-) adipocytes (Fig. 4)
. Consistent with the reduced
IRS-1 phosphorylation, insulin-stimulated, IRS-1-associated PI3 kinase
activity was reduced in both N/H and H/H adipocytes (Fig. 5)
. Since the
expression of IR was not changed in N/H adipocytes, the data suggested
that IR tyrosine kinase activity is susceptible to changes in
circulating insulin concentration. Chronic hyperglycemia, together with
hyperinsulinemia, leads to the down-regulation of IR expression and the
further reduction of insulin-stimulated protein tyrosine
phosphorylation in H/H GLUT4(+/-) adipocytes (Figs. 3
, 4)
.
Tyrosine-phosphorylated IRS-1 and pp60/IRS-3 serve as docking proteins
for the binding of other proteins such as the p85 subunit of PI3 kinase
(9
, 29
, 31)
. Binding of p85 to IRS-1 and/or pp60/IRS-3
activates PI3 kinase (9
, 29
, 31)
. It has been suggested
that PI3 kinase is involved in mediating insulin-regulated glucose
metabolism (32
, 33)
. Decreased IRS-1 tyrosine
phosphorylation was accompanied by reduced insulin-stimulated,
IRS-1-associated PI3 kinase activity in N/H and H/H GLUT4(+/-)
adipocytes (Fig. 5)
. However, expression of p85 protein was increased
by 80% in diabetic H/H GLUT4(+/-) adipocytes (Fig. 4)
. Along with
increased p85 expression, p85-associated PI3 kinase activity was
increased by 50% in the basal state and insulin-stimulated,
p85-associated PI3 kinase activity was not altered in H/H GLUT4(+/-)
adipocytes compared to controls (Fig. 6)
. Although the mechanism of p85
protein up-regulation is unknown, the normal insulin-stimulated total
p85 activity suggests that other pathways are activated and compensate
for the reduction in IRS-1-associated PI3 kinase activity in H/H
adipocytes. Such unchanged insulin-stimulated total PI3 kinase activity
might be responsible for the absence of further reduction in glucose
uptake in H/H adipocytes.
The reduction of GLUT4 expression was similar among N/N, N/H, and H/H
adipocytes of GLUT4(+/-) mice (Fig. 1)
. In addition, maximal
insulin-stimulated glucose uptake was similar in all three groups of
GLUT4(+/-) adipocytes (Fig. 2)
. The reduction in insulin-stimulated
glucose uptake was similar to the decrease in GLUT4 protein content.
The progressive and severe reduction of protein tyrosine
phosphorylation and the decrease in insulin-stimulated,
IRS-1-associated PI3 kinase activity in N/H and H/H adipocytes did not
cause further reduction in maximal insulin-stimulated glucose uptake.
However, glucose uptake at low insulin concentrations (1 nM in N/H and
5 nM in H/H) in N/H and H/H adipocytes of GLUT4(+/-) was significantly
reduced compared to controls. On the other hand, a normal stimulation
of glucose uptake was observed in N/N GLUT4(+/-) adipocytes at an
insulin concentration of 1 nM (Fig. 2A
), which is consistent
with the normal insulin-stimulated glu protein tyrosine phosphorylation
in N/N adipocytes. Thus, it is clear that cellular GLUT4 content is the
rate-limiting factor in determining maximal insulin-stimulated glucose
uptake in GLUT4(+/-) adipocytes. However, the N/N adipocytes have
normal glucose uptake at physiological insulin levels, with only half
the normal amount of GLUT4. This demonstrates that adipocytes with
normal insulin signaling can maintain normal glucose uptake even with a
severely reduced GLUT4 content.
The lack of effect of reduced IRS-1-associated PI3 kinase activity on
maximal insulin-stimulated glucose uptake in GLUT4(+/-) adipocytes was
also observed in adipocytes of aged gold thioglucose-induced obese mice
and 3T3 L1 adipocytes (17
, 34
35
36)
. One explanation could
be that low levels of insulin-stimulated, IRS-1-associated PI3 kinase
activity are sufficient to fully stimulate GLUT4 mediated glucose
transport in H/H GLUT4(+/-) adipocytes. Early studies in human
adipocytes suggested that only a small portion of IRS-1 phosphorylation
is required for full stimulation of glucose uptake (19)
.
In addition, the extent to which IRS-1-associated PI3 kinase activity
was reduced was not proportional to the reduction of IRS-1 tyrosine
phosphorylation. It also suggested that in the insulin signaling
cascade, protein is phosphorylated in excess to that required for the
stimulation of the next step.
Another possibility that has been proposed is that IRS-1-independent
pathways may lead to the activation of PI3 kinase, GLUT4 translocation
and glucose uptake (34
35
36)
. This is supported by the
observation that total insulin-stimulated PI3 kinase activity was
normal in H/H GLUT4(+/-) adipocytes. Conflicting results exist for the
involvement of IRS-1 in insulin-stimulated GLUT4 translocation and
glucose uptake. Insulin-activated, IRS-1-associated PI3 kinase was
identified in GLUT4-containing vesicles (37)
, whereas
microinjection of antibodies against IRS-1 did not block
insulin-stimulated, GLUT4-containing vesicle translocation
(38)
. Recent studies of 3T3 L1 adipocytes as well as
adipocytes from NIDDM patients suggested that other mechanisms that
activate PI3 kinase, such as through docking to IRS-2, could play an
important role in insulin-stimulated adipocytes glucose uptake
(22
, 34
35
36)
. Thus, increased IRS-2 or pp60/IRS-3
expression or docking of PI3 kinase to these proteins could be the
potential mechanism for the maintenance of insulin-stimulated PI3
kinase activity in H/H GLUT4(+/-) adipocytes. The maintenance of
insulin-stimulated PI3 kinase activity in H/H GLUT4(+/-) adipocytes
appears to protect cells from further reductions in glucose uptake.
In summary, these experiments represent the first longitudinal study describing changes in the insulin signaling pathway that occur in adipocytes from normal to an insulin-resistant state. Changes in insulin signaling coincide with the development of circulating hyperinsulinemia. Hyperglycemia and hyperinsulinemia ultimately lead to decreased IR protein expression, decreased IRS-1 and pp60/IRS-3 tyrosine phosphorylations, and decreased IRS-1-associated PI3 kinase activation. Expression of p85 protein is up-regulated and total insulin-stimulated PI3 kinase activity remains normal in H/H GLUT4(+/-) adipocytes, suggesting a compensatory response to the reduced GLUT4 expression and insulin signaling. Despite the impaired insulin signaling in N/H and H/H adipocytes, maximal insulin-stimulated glucose uptake is not further reduced, demonstrating that the cellular GLUT4 content is the rate-limiting factor in mediating maximal insulin-stimulated glucose uptake in adipocytes. Finally, the experiments described above also show that the insulin signaling cascade has sufficient plasticity to accommodate significant changes in specific components without further reducing glucose uptake.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  D. P. Sparling, B. A. Griesel, and A. L. Olson Hyperphosphorylation of MEF2A in primary adipocytes correlates with downregulation of human GLUT4 gene promoter activity Am J Physiol Endocrinol Metab, April 1, 2007; 292(4): E1149 - E1156. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Li, M. Raghunath, D. Tan, R. R. Lareu, Z. Chen, and R. W. Beuerman Defensins HNP1 and HBD2 Stimulation of Wound-Associated Responses in Human Conjunctival Fibroblasts. Invest. Ophthalmol. Vis. Sci., September 1, 2006; 47(9): 3811 - 3819. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. McClain, M. Hazel, G. Parker, and R. C. Cooksey Adipocytes with increased hexosamine flux exhibit insulin resistance, increased glucose uptake, and increased synthesis and storage of lipid Am J Physiol Endocrinol Metab, May 1, 2005; 288(5): E973 - E979. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. T. Fueger, H. S. Hess, K. A. Posey, D. P. Bracy, R. R. Pencek, M. J. Charron, and D. H. Wasserman Control of Exercise-stimulated Muscle Glucose Uptake by GLUT4 Is Dependent on Glucose Phosphorylation Capacity in the Conscious Mouse J. Biol. Chem., December 3, 2004; 279(49): 50956 - 50961. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
