Content uploaded by Richard Sciotti
Author content
All content in this area was uploaded by Richard Sciotti on Nov 19, 2015
Content may be subject to copyright.
Enhanced Basal Activation of Mitogen-Activated
Protein Kinases in Adipocytes From Type 2 Diabetes
Potential Role of p38 in the Downregulation of GLUT4
Expression
Christian J. Carlson,
1
Sandra Koterski,
1
Richard J. Sciotti,
1
German Braillard Poccard,
2
and Cristina M. Rondinone
1
Serine and threonine kinases may contribute to insulin
resistance and the development of type 2 diabetes. To
test the potential for members of the mitogen-activated
protein (MAP) kinase family to contribute to type 2
diabetes, we examined basal and insulin-stimulated Erk
1/2, JNK, and p38 phosphorylation in adipocytes iso-
lated from healthy and type 2 diabetic individuals.
Maximal insulin stimulation increased the phosphoryla-
tion of Erk 1/2 and JNK in healthy control subjects but
not type 2 diabetic patients. Insulin stimulation did not
increase p38 phosphorylation in either healthy control
subjects or type 2 diabetic patients. In type 2 diabetic
adipocytes, the basal phosphorylation status of these
MAP kinases was significantly elevated and was associ-
ated with decreased IRS-1 and GLUT4 in these fat cells.
To determine whether MAP kinases were involved in the
downregulation of IRS-1 and GLUT4 protein levels,
selective inhibitors were used to inhibit these MAP
kinases in 3T3-L1 adipocytes treated chronically with
insulin. Inhibition of Erk 1/2, JNK, or p38 had no effect
on insulin-stimulated reduction of IRS-1 protein levels.
However, inhibition of the p38 pathway prevented the
insulin-stimulated decrease in GLUT4 protein levels. In
summary, type 2 diabetes is associated with an in-
creased basal activation of the MAP kinase family.
Furthermore, upregulation of the p38 pathway might
contribute to the loss of GLUT4 expression observed in
adipose tissue from type 2 diabetic patients. Diabetes
52:634 – 641, 2003
The number of individuals afflicted with diabetes
worldwide is estimated to double by 2025 (1 and
references therein). Of these cases, the vast
majority will be type 2 diabetes (1). Insulin
resistance, the impaired ability for tissues to respond to
insulin, may be the predominant defect leading to the
development of impaired glucose tolerance and ultimately
type 2 diabetes (1). The underlying cellular mechanisms
leading to insulin resistance are unknown but are believed
to be due to defects in insulin signal transduction and not
to defects in the insulin receptor itself or insulin:insulin
receptor binding.
IRS-1 plays a key role in insulin signal transduction, and
GLUT4 is necessary for insulin-stimulated glucose uptake
(2–5). IRS-1 and GLUT4 protein levels are diminished in
the adipocytes of patients with type 2 diabetes (6 –9). IRS-1
and GLUT4 protein levels are also decreased in the adipo-
cytes from first-degree relatives of patients with type 2
diabetes (8). Experiments in transgenic mice have demon-
strated the importance of these proteins in maintaining
insulin sensitivity. Decreased GLUT4 expression, as either
a heterozygous knockout or a GLUT4 gene ablation spe-
cifically in adipose tissue, leads to whole-body insulin
resistance and eventually diabetes (4,5). Adipocytes iso-
lated from IRS-1 knockout mice have a decreased insulin-
stimulated rate of glucose uptake and diminished
translocation of GLUT4 to the plasma membrane (2,3).
Thus the loss of these proteins, specifically in adipose
tissue, may be a significant contributor to the onset of
insulin resistance and development of diabetes.
Serine and threonine kinases are believed to play an
important role in the onset of insulin resistance by regu-
lating IRS-1 and GLUT4 protein levels through both pre-
and posttranslational mechanisms (6,10). Phosphorylation
of IRS-1 on serine and/or threonine residues precedes the
molecules’ subsequent proteasomal degradation in insulin-
resistant 3T3-L1 adipocytes (10,11). Not unexpectedly,
insulin resistance in cells and animals is associated with
increased serine kinase activity toward IRS-1 (12). Fur-
thermore, serine/threonine kinases are often involved with
the regulation of gene expression by serving as intermedi-
ates in signal transduction cascades that link extracellular
and intracellular stimuli to the nucleus (13). Thus serine/
From
1
Insulin Signaling, Metabolic Diseases Division, Global Pharmaceutical
Products Division, Abbott Laboratories, Abbott Park, Illinois; and the
2
Unit of
Endocrinology and Diabetes, University Hospital San Martin, Corrientes,
Argentina.
Address correspondence and reprint requests to Cristina M. Rondinone,
Insulin Signaling, Metabolic Diseases Division, Global Pharmaceutical Prod-
ucts Division, Abbott Laboratories, Abbott Park, IL 60064. E-mail: cristina.
rondinone@abbott.com.
Received for publication 1 May 2002 and accepted in revised form 6
December 2002.
C/EBP, CCAAT-enhancer binding protein; ECL, enhanced chemilumines-
cence; IC
50
, half-maximal inhibitory concentration; JNK, Jun kinase; MAP,
mitogen-activated protein; NF-1, nuclear factor 1; PI3, phosphatidylinositol 3;
TNF, tumor necrosis factor.
634 DIABETES, VOL. 52, MARCH 2003
threonine kinases have the potential to contribute to the
diminished GLUT4 mRNA levels in insulin-resistant adi-
pose tissue.
Specifically, members of the mitogen-activated protein
(MAP) kinase family of serine-threonine kinases may
contribute to the development of insulin resistance. IRS-1
contains many potential MAP kinase phosphorylation sites
(14), and it has been observed that certain MAP kinases
are capable of phosphorylating IRS-1 (12,15,16). MAP
kinases play important roles in the regulation of gene
expression through the phosphorylation-mediated activa-
tion/inactivation of numerous transcription factors (13).
Thus MAP kinases are theoretically capable of regulating
IRS-1 and GLUT4 protein levels through pre- and post-
translational mechanisms. Furthermore, insulin stimula-
tion is capable of activating members of the MAP kinase
family, specifically Erk 1/2, JNK, and p38, in various cell
types (17–21). In human skeletal muscle, insulin signaling
to Erk 1/2 is normal from patients with type 2 diabetes
compared with healthy control subjects, despite a dimin-
ished insulin responsiveness of components required for
insulin’s metabolic effects (i.e., the phosphatidylinositol 3
[PI3] kinase and protein kinase B/AKT pathway) (22,23).
However, the activation state and insulin responsiveness
of the MAP kinases in adipose tissue from type 2 diabetic
patients is unknown.
Herein, we compare and contrast the ability of insulin to
stimulate the phosphorylation of the MAP kinases in
isolated adipocytes from healthy individuals and patients
with type 2 diabetes. An increased basal level of phosphor-
ylation of Erk 1/2, JNK, or p38 was observed in adipocytes
from type 2 diabetic subjects in association with a reduc-
tion in IRS-1 and GLUT4, suggesting that elevated MAP
kinase activation may contribute to the type 2 diabetic
condition. Furthermore, selective inhibitors of the p38
pathway, but not the Erk 1/2 or JNK pathway, attenuated
the decrease in GLUT4 protein abundance in response to
chronic insulin exposure of 3T3-L1 adipocytes, without
affecting the insulin-stimulated loss of IRS-1. Taken to-
gether, these results—the increased phosphorylation of
p38 in human adipocytes from type 2 diabetic subjects and
the necessary role of p38 in insulin-stimulated downregu-
lation of GLUT4 —suggest that this kinase may play a key
role in the development of insulin resistance and type 2
diabetes.
RESEARCH DESIGN AND METHODS
Human subjects. Specimens of human subcutaneous adipose tissue were
obtained from the abdominal region of nondiabetic and type 2 diabetic
subjects (Table 1). Subjects were recruited from the Unit of Endocrinology
and Diabetes at University Hospital San Martin (Corrientes, Argentina) and
Northwestern University Medical School (Chicago, IL). Subjects signed an
informed consent document indicating that they understood the risks and
benefits of participation in this experiment. The experimental procedures
were approved by the appropriate scientific review committees at University
Hospital Corrientes and Northwestern University Medical School.
Isolation and preparation of human adipocytes. Adipose cells were
prepared as described previously (7,24). Briefly, the adipocytes were isolated
by digesting ⬃0.6 g tissue for 50 min at 37°C in medium 199 containing 25
mmol/l HEPES, 4% BSA, and 5.5 mmol/l glucose and collagenase (Sigma) at 0.8
mg/ml in a shaking water bath. Adipocyte cell size and number were
determined as described previously (24). Isolated human adipocytes were
distributed into plastic vials (12–15% cell suspension) in a final incubation
volume of 400 l. Cells were preincubated with or without 6.9 nmol/l insulin
for 15 min, immediately separated by centrifugation through silicon oil, lysed
in 0.4 ml lysis buffer containing 25 mmol/l Tris-HCl (pH 7.4), 0.5 mmol/l EGTA,
25 mmol/l NaCl, 1% Nonidet P-40, 1 mmol/l Na
3
VO
4
, 10 mmol/l NaF, 0.2 mmol/l
leupeptin, 1 mmol/l benzamidine, and 0.1 mmol/l 4-(2-aminoethyl)benzenesul-
fonyl fluoride hydrochloride, and rocked for 40 min at 4°C. Detergent-
insoluble material was sedimented by centrifugation at 12,000gfor 10 min at
4°C. Protein concentration of the cell lysates was determined by bicinchoninic
acid assay (Pierce Chemical, Rockford, IL). This insulin concentration (6.9
nmol/l) has been previously determined to achieve maximal insulin stimula-
tion of human adipocytes (7).
Immunoblotting. To minimize any researcher bias, the researchers were
blinded to the identity of the subjects from whom samples were taken. Total
protein (10 g) was separated on 10% acrylamide SDS-PAGE gels (BioRad)
and transferred to nitrocellulose (Millipore). Membranes were blocked in 4%
nonfat dry milk in TBS-T (Tris-buffered saline with 0.5% Tween-20) (Sigma),
and primary antibodies were diluted in either 4% nonfat dry milk/TBS-T or
0.1% BSA/TBS-T. The blots were probed with various primary antibodies:
phospho-specific antibodies toward Erk 1/2 and Jun kinase (JNK) (Promega,
Madison, WI) phospho-specific antibodies to p38 MAP kinase (Cell Signaling
Technologies, Beverly, MA); Erk 1/2, JNK, and p38 independent of phosphor-
ylation state (Upstate Biotechnology, Lake Placid, NY; Santa Cruz Biotech-
nology, Santa Cruz, CA; and Cell Signaling Technologies, respectively); IRS-1,
IRS-2, and the p85 regulatory subunit of PI3 kinase (whole antiserum)
(Upstate Biotechnology); and GLUT4 (Chemicon, Temecula, CA). Membranes
were washed thoroughly and incubated with donkey anti-rabbit antibodies
conjugated to horseradish peroxidase (Amersham). After washing, protein
bands were visualized using enhanced chemiluminescence (ECL) (Amer-
sham) and exposure to Hyperfilm ECL X-ray film (Amersham). Densitometric
analysis of the bands was performed using a laser densitometer (PDSI;
Amersham) and analyzed by ImageQuant software (Amersham).
p38 and JNK in vitro kinase assays. The half-maximal inhibitory concen-
tration (IC
50
) toward p38 and JNK of the compounds used in this experiment
were determined using in vitro kinase assays. The new compounds (A-291077
and A-304000) are aza-azulene–based compounds designed to inhibit p38 MAP
kinase. Recombinant p38 (␣) or JNK were activated by incubation at 30°C for
1 h with recombinant MKK6 (4 mU) in 25 mmol/l HEPES (pH 7.4), 25 mmol/l
-glycerophosphate, 1 mmol/l Na
3
VO
4
, 1 mmol/l DTT, 25 mmol/l MgCl
2
,5
mmol/l EDTA, 1 mmol/l NaF, and 50 mol/l ATP. After activation of the
kinase, activated p38 was incubated with 8 g recombinant TRDH-ATF2 in 25
mmol/l HEPES (pH 7.4), 25 mmol/l -glycerophosphate, 1 mmol/l Na
3
VO
4
,1
mmol/l dithiothreitol (DTT), 25 mmol/l MgCl
2
, 5 mmol/l EDTA, and 1 mmol/l
NaF. Reactions were started with the addition of [
33
P]ATP (10 mol/l final;
Amersham). Kinase reactions were allowed to proceed at 30°C for 1 h, after
which the reactions were stopped with 5% phosphoric acid. The samples were
transferred to filtration plates prewetted with 1% phosphoric acid and
incubated for 15 min at room temperature. Vacuum filter plates were washed
three times each with 1% phosphoric acid and counted in Microscint-20 on a
Packard Topcount instrument.
3T3-L1 cell culture. 3T3-L1 cells were grown and maintained as fibroblasts
in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Gaith-
ersburg, MD) with high glucose containing 10% FBS (HyClone, Logan, UT) in
a humidified atmosphere composed of 95% air and 5% CO
2
. Cells were
differentiated into adipocytes by exposure to DMEM high glucose with 10%
FBS, 0.4 g/ml dexamethasone, 0.5 mmol/l isobutylmethylxanthine, and 5
g/ml insulin. After 3 days, the medium was changed and the cells were
maintained with DMEM containing 10% FBS. Day 10 adipocytes were starved
for2hinDMEM/0.1% FBS before incubation in DMEM/0.1% FBS with or
without insulin (1 mol/l) for the time specified. In some cases, the cells were
pretreated for 30 min before exposure to insulin with various reagents
including SB203580, PD95085, A-291077, and A-304000. At the appropriate
time, medium was aspirated from the cells and the cells were washed with
ice-cold PBS. Subsequently, cells were lysed in lysis buffer [25 mmol/l Tris-HCl
(pH 7.4), 0.5 mmol/l EGTA, 25 mmol/l NaCl, 1% Nonidet P-40, 1 mmol/l
Na
3
VO
4
, 10 mmol/l NaF, 0.2 mmol/l leupeptin, 1 mmol/l benzamidine, and 0.1
TABLE 1
Characteristics of subjects
Healthy Type 2 diabetes
n(M, F) 7 (6, 1) 7 (4, 3)
Age (years) 40.3 ⫾11.77 44.6 ⫾17.6
BMI (kg/m
2
)26 ⫾5.42 33 ⫾3.37*
Triglyceride content (g/cell) 0.37 ⫾0.107 0.57 ⫾0.169
Blood glucose (mg/dl) 84.17 ⫾10.13 202.33 ⫾60.79*
Blood insulin (U/ml) 8.12 ⫾2.27 25.15 ⫾15.23*
Data are means ⫾SD. *Significant difference from healthy (P⬍0.05).
C.J. CARLSON AND ASSOCIATES
DIABETES, VOL. 52, MARCH 2003 635
mmol/l 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride] (Calbio-
chem, La Jolla, CA) and rocked for 30 min at 4°C. Detergent-insoluble material
was sedimented by centrifugation at 12,000gfor 10 min at 4°C. Protein
concentration of the cell lysates was determined by bicinchoninic acid assay
(Pierce Chemical). Protein (20 g) was separated on 7.5% SDS-PAGE gels
(BioRad, Hercules, CA) and transferred to nitrocellulose. Western blotting
was performed as described above.
Statistical analysis. Data are presented as means ⫾SD. Statistical signifi-
cance was set at P⬍0.05. Statistically significant differences in subject
characteristics between healthy control subjects and type 2 diabetic patients
were detected with an unpaired Student’s two-tailed ttest. Differences in
phosphorylation status of the MAP kinases in the basal and insulin-stimulated
state, as well as between healthy and type 2 diabetic patients, were deter-
mined using repeated measures ANOVA (JMP, version 3.1; SAS Institute, Cary,
NC). A least significant difference test was used for posthoc analysis.
RESULTS
Patient characteristics. The type 2 diabetic patients
examined in this study fit many of the previously described
characteristics associated with obesity and type 2 diabe-
tes, including BMI, blood glucose, and insulin concentra-
tions (1,6 –9). Type 2 diabetic patients had significantly
greater blood glucose and blood insulin concentrations
compared with healthy control subjects (P⬍0.05) (Table
1). Although the difference was not statistically significant
(P⫽0.06), there was a tendency for adipocyte size to be
greater in type 2 diabetic patients. BMI was greater in
patients with type 2 diabetes compared with healthy
control subjects. Because the control subjects were not
matched for BMI, the following experiments cannot differ-
entiate between the effects of obesity and type 2 diabetes
on the parameters examined.
IRS-1 and GLUT4 protein abundances are decreased
in adipocytes isolated from patients with type 2
diabetes. Insulin resistance in adipocytes is frequently
characterized by diminished protein abundances of IRS-1
and GLUT4 (2–9). In the current report (Fig. 1), IRS-1 and
GLUT4 protein abundances are decreased 70% and 50%,
respectively, in adipocytes from type 2 diabetic patients
compared with healthy control subjects. This decrement is
similar in magnitude to previously reported comparisons
of IRS-1 and GLUT4 protein abundances in adipocytes
from type 2 diabetic patients and healthy control subjects
(6 –9).
Basal MAP kinase phosphorylation is increased in
adipocytes isolated from patients with type 2 diabe-
tes. In adipocytes from healthy humans, 15 min of maxi-
mal insulin stimulation resulted in 1.8- and 2.0-fold
elevated phosphorylation of Erk 1/2 and JNK, respectively
(Fig. 2). p38 phosphorylation in response to insulin was
observed in only two of seven healthy subjects examined.
Interestingly, insulin treatment resulted in the phosphory-
lation of only a subset of JNK isoforms, specifically the p46
isoforms in both human adipocytes and 3T3-L1 adipocytes
(data not shown). Although previous experiments have
demonstrated JNK to be activated in response to insulin
(17,18,20), this is the first report of an isoform-specific
FIG. 1. IRS-1 and GLUT4 protein abundances are decreased in adipo-
cytes isolated from patients with type 2 diabetes. Human adipocytes
were isolated as described in RESEARCH DESIGN AND METHODS and stimu-
lated with 6.9 nmol/l insulin for 15 min. Cells were lysed, and total
protein was subjected to 10% SDS-PAGE and immunoblotted with the
indicated antibodies. Immunoblots were quantified using laser densi-
tometry and are reported as integrated optical densities in arbitrary
units. The representative immunoblots show data from one healthy
individual and one patient with type 2 diabetes (T2D). The data
presented are means and SDs for seven healthy and seven type 2
diabetic patients (see Table 1 for characteristics). #Significant differ-
ence from healthy (P<0.05).
FIG. 2. MAP kinase phosphorylation status in insulin-stimulated adi-
pocytes from healthy humans and patients with type 2 diabetes. Human
adipocytes were isolated as described in RESEARCH DESIGN AND METHODS
and stimulated with 6.9 nmol/l insulin for 15 min. Cells were lysed, and
total protein was subjected to 10% SDS-PAGE and immunoblotted with
the indicated antibodies [Erk 1/2 (A), JNK (B), and p38 (C)]. Immu-
noblots were quantified using laser densitometry and are reported as
integrated optical densities in arbitrary units. The representative
immunoblots show data from one healthy individual and one patient
with type 2 diabetes (T2D). The data presented are means and SDs for
seven healthy and seven type 2 diabetic patients (see Table 1 for
characteristics). #Significant difference from healthy (P<0.05); *sig-
nificant difference from the corresponding basal value (P<0.05).
MAP KINASES AND TYPE 2 DIABETES
636 DIABETES, VOL. 52, MARCH 2003
response. Furthermore, this is the first report to describe
the basal phosphorylation status and insulin responsive-
ness of members of the MAP kinase family in human
adipocytes.
In contrast to healthy adipocytes, Erk 1/2 and JNK
phosphorylation was not increased by insulin in type 2
diabetic adipocytes. The failure of insulin to increase MAP
kinase phosphorylation may have been due to an elevated
basal level of phosphorylation, which was significantly
greater than in untreated healthy adipocytes. The most
intriguing result, however, was the profound fivefold in-
crease in p38 phosphorylation observed in the basal state
of type 2 diabetic adipocytes. Insulin stimulation did not
further increase p38 phosphorylation in type 2 diabetic
adipocytes. The protein abundances of Erk 1/2, JNK, and
p38 were not significantly different between healthy con-
trol subjects and patients with type 2 diabetes.
MAP kinase inhibition has no effect on insulin-stim-
ulated loss of IRS-1 protein abundance, but p38 is
necessary for the loss of GLUT4. To gain further insight
into the potential mechanisms by which basal activation of
MAP kinases in adipocytes may contribute to the develop-
ment of insulin resistance, we systematically tested the
role of Erk 1/2, JNK, and p38 using a panel of specific
inhibitors in insulin-resistant 3T3-L1 adipocytes. 3T3-L1
adipocytes can be made insulin resistant through chronic
exposure to insulin and elevated glucose concentrations
(25–27). This model of insulin resistance mimics the
expression pattern of human adipocytes in that there is
a decreased protein abundance of IRS-1 and GLUT4
(10,11,25–27).
The panel of inhibitors used in this experiment includ-
ed the MEK inhibitor PD98059 (to inhibit the Erk 1/2
pathway) as well as the p38 inhibitor SB203580 (28). In
addition, we used two new compounds (Table 2) that
show inhibitory properties toward p38 and JNK (A-291077)
or p38-alone (A-304000) (29). Table 2 presents the chemi-
cal structures and IC
50
toward p38 and JNK as determined
with in vitro kinase assays. The compound A-304000 is a
selective inhibitor of p38, with little inhibitory activity
toward JNK. Furthermore, these new compounds, specif-
ically A-304000, exhibited minimal inhibitory activity in in
vitro kinase reactions using AKT/PKB, PKA, MAPKAP2,
casein kinase2, PKC␥, AMP kinase, c-Met, IGF receptor,
Polo-like kinase, and LCK kinase. Thus the pattern of
inhibition achieved by this panel would test the contribu-
tion of Erk 1/2 (PD98059), JNK, and p38 (A-291077) and
p38-alone (A-304000) in the downregulation of GLUT4
expression and/or IRS-1 protein abundance in a cell cul-
ture model of insulin resistance. The overlapping specific-
ity of A-291077 and A-304000 would allow us to infer the
role that either JNK or p38 may play in the insulin-
stimulated loss of IRS-1 and/or GLUT4.
Chronic exposure to insulin in a high-glucose environ-
ment significantly decreased protein levels of IRS-1 and
GLUT4, by 50% and 40%, respectively (Fig. 3). This effect
was restricted to IRS-1 and GLUT4, as insulin exposure
had no effect on the levels of p85 PI3 kinase or IRS-2 (data
not shown) as previously reported (11). Treatment of the
3T3-L1 adipocytes with selective inhibitors of Erk 1/2,
JNK, or p38 did not prevent the insulin-stimulated loss of
IRS-1.
The insulin-stimulated loss of GLUT4 protein abundance
was prevented only by inhibitors of p38 (Fig. 3). Chronic
exposure of the 3T3-L1 adipocytes to insulin resulted in a
significant decrease in GLUT4 protein levels. Treatment of
the 3T3-L1 adipocytes with p38 inhibitors without insulin
stimulation did not significantly alter the protein abun-
dance of GLUT4. Treatment with 10 mol/l of the p38
selective inhibitors SB203580 or A-304000 did not prevent
the insulin-stimulated downregulation of GLUT4. How-
ever, 50 mol/l of SB203580 or A-304000 resulted in a
GLUT4 protein level that was not statistically different
from that of untreated/unstimulated cells or cells treated
with 50 mol/l of compound without insulin. These data
indicate that inhibition of p38 is sufficient to prevent the
insulin-stimulated downregulation of GLUT4. Pretreat-
ment of the 3T3-L1 adipocytes with A-291077, an inhibi-
tor of both JNK and p38, resulted in attenuation of the
insulin-stimulated loss of GLUT4. This effect was observed
at 10- and 50-mol/l concentrations of compound. By
contrast, inhibition of the Erk 1/2 pathway with the MEK
inhibitor PD98059 had no effect on the insulin-stimulated
loss of GLUT4 protein.
A-304000 prevented the insulin-induced loss of GLUT4
protein abundance in a dose-responsive manner (Fig. 4),
with a ⬎40% recovery of GLUT4 protein abundance ob-
served with 25 mol/l and a full recovery at 50 mol/l.
Taken together, these results indicate that inhibition of
p38, but not Erk 1/2, can prevent the insulin-induced
downregulation of GLUT4.
p38 phosphorylation is transiently increased by insu-
lin in 3T3-L1 adipocytes. Insulin stimulation resulted in
an increased p38 phosphorylation by approximately two-
fold at 5 and 10 min, before returning to baseline for the
remainder of the experiment (Fig. 5A). Inhibition of p38
with A-304000 (Fig. 5B) or SB203580 (data not shown)
prevented the insulin-induced p38 phosphorylation at 5
TABLE 2
IC
50
(in mol/l) calculated from in vitro kinase assays
A-304000 A-291007
p38 4.86 0.64
JNK ⬎50 3.47
Casein kinase 2 ⬎50 ⬎50
AKT 1 ⬎50 ⬎50
PKA ⬎50 7.3
PKC ␥⬎50 ⬎50
MAPKAP 2 ⬎50 ⬎50
AMP kinase ⬎50 ⬎50
c-Met ⬎50 ⬎50
IGF receptor ⬎50 ⬎50
Polo-like kinase ⬎50 ⬎50
LCK kinase ⬎50 ⬎50
C.J. CARLSON AND ASSOCIATES
DIABETES, VOL. 52, MARCH 2003 637
and 10 min of insulin stimulation. p38 protein levels were
unaffected by insulin stimulation or the inhibitors.
DISCUSSION
This report is the first to describe the phosphorylation
status and insulin responsiveness of the MAP kinases in
human adipocytes from healthy control subjects and pa-
tients with type 2 diabetes. Members of the MAP kinase
family have the potential to contribute to the perturbations
in insulin signaling associated with type 2 diabetes. Thus
we hypothesized that if one or more of the MAP kinases
examined were involved with type 2 diabetes, there should
be differential phosphorylation status of that particular
MAP kinase between healthy adipocytes and type 2 dia-
betic adipocytes. In this report, we have demonstrated that
basal MAP kinase phosphorylation, especially that of p38,
is elevated in adipocytes from type 2 diabetic patients, as
in previous experiments that have found elevated serine
kinase activity in adipocytes from insulin-resistant animals
(12), and our results are consistent with MAP kinases
contributing to type 2 diabetes. The increased basal phos-
phorylation was most striking for p38, which exhibited a
fivefold increase in adipocytes from type 2 diabetic pa-
tients compared with healthy control subjects. In contrast,
Erk 1/2 and JNK phosphorylation was increased approxi-
mately twofold in adipocytes from type 2 diabetic patients
compared with healthy control subjects.
The elevated basal phosphorylation state of members of
the MAP kinase family in adipocytes from type 2 diabetic
subjects is associated with a reduction in IRS-1 and GLUT4
protein levels. In humans, diminished GLUT4 and IRS-1
protein abundance can predict the future development of
type 2 diabetes (8,9), suggesting that dysregulation of the
expression of these proteins may be an early step toward
FIG. 3. MAP kinase inhibition has no effect on insulin-stim-
ulated loss of IRS-1 protein abundance, but p38 is necessary
for the loss of GLUT4. 3T3-L1 adipocytes were pretreated
with 10 or 50 mol/l SB20358 (A), A-304000 (B), A-291077
(C), or PD98059 (D) before stimulation with 1 mol/l insulin
overnight. Cells were lysed, and total protein was subjected
to 7.5% SDS-PAGE and immunoblotted with the indicated
antibodies. Immunoblots were quantified using laser densi-
tometry and are reported as integrated optical densities in
arbitrary units. Representative Western blots are shown for
GLUT4, IRS-1, and p85. The corresponding bar graphs pres-
ent the results as means and SDs of at least three indepen-
dent experiments for GLUT4 and IRS-1 as indicated. The
x-axis represents (from left to right) unstimulated cells
(lane 1), cells treated with insulin overnight (lane 2), and
cells pretreated with compound without or with insulin (lanes
3– 6). The y-axis represents at least three independent ob-
servations expressed as arbitrary units. *Significant differ-
ence from the corresponding basal value (P<0.05).
FIG. 4. Inhibition of p38 with A-304000 prevents the insulin-stimulated
loss of GLUT4 protein abundance in a dose-responsive manner. 3T3-L1
adipocytes were pretreated with A-304000 at the indicated concentra-
tions, before stimulation with 1 mol/l insulin overnight. Cells were
lysed, and total protein was subjected to 7.5% SDS-PAGE and immu-
noblotted with the indicated antibodies. Immunoblots were quantified
using laser densitometry and are reported as integrated optical densi-
ties in arbitrary units. The data are presented as means and SDs from
at least three independent experiments.
MAP KINASES AND TYPE 2 DIABETES
638 DIABETES, VOL. 52, MARCH 2003
the development of type 2 diabetes. To begin to under-
stand the mechanism by which activation of the MAP
kinases might contribute to insulin resistance and type 2
diabetes, we systematically tested the role of these path-
ways in regulating IRS-1 and GLUT4 protein levels in a
cell-culture model of insulin resistance. We used a panel of
inhibitors that selectively inhibited Erk 1/2 (PD98059), p38
alone (A-304000), and p38 and JNK (A-291077) to test the
contribution of all three MAP kinase pathways toward
insulin-stimulated downregulation of IRS-1 and GLUT4.
The insulin-stimulated loss of IRS-1 was not prevented by
inhibition of any of the MAP kinase pathways examined,
consistent with a prior publication that demonstrated that
PD98059 has no effect on the insulin-stimulated loss of
IRS-1 (11). These results expand on previous findings to
rule out JNK and p38 as playing a role in IRS-1 degrada-
tion. Inhibition of mTOR with rapamycin has been previ-
ously shown to prevent serine phosphorylation and
subsequent degradation of IRS-1 (11). Although these
results indicate that these MAP kinases do not contribute
to insulin-induced IRS-1 degradation, we cannot rule out
the possibility that they may be involved with serine
phosphorylation of IRS-1, potentially leading to the devel-
opment of insulin resistance.
The insulin-induced loss of GLUT4 protein abundance
could be prevented by selective inhibitors of p38. This
effect was also observed when both p38 and JNK were
inhibited by A-291077, whereas inhibition of the Erk 1/2
pathway had no effect. Because the inhibition of both p38
and JNK simultaneously prevented the insulin-stimulated
downregulation of GLUT4 in a manner similar to inhibition
of p38 alone, we conclude that p38 is a major contributor
to the insulin-stimulated loss of GLUT4. Although we
cannot completely rule out the possibility that JNK and/or
other kinases may be involved, our results are consistent
with the findings of Fujishiro et al. (30), who demonstrated
that constitutive activation of the MKK6-p38 pathway, but
not the MKK7-JNK pathway, was sufficient for the down-
regulation of GLUT4 expression.
The role of p38 in the insulin-stimulated downregulation
of GLUT4 is further supported by our use of two structur-
ally distinct inhibitors, SB203580 and A-304000. These
selective p38 inhibitors prevented the insulin-stimulated
downregulation of GLUT4 with similar potencies. Al-
though treatment with 50 mol/l SB203580 resulted in
decreased protein levels of IRS-1, IRS-2, and p85 regard-
less of insulin stimulation, treatment did not decrease
GLUT4 protein levels and prevented the insulin-stimulated
loss of GLUT4. This suggests that high doses of SB203580
may be toxic to the cells but selectively preserve GLUT4
expression, consistent with p38 acting as a negative regu-
lator of GLUT4 expression. These cytotoxic effects appear
to be limited to SB203580 itself and are not related to
inhibition of p38 per se, because the other p38 inhibitors
(A-304000 and A-291077) did not affect IRS-1, p85, or IRS-2
levels. Thus, our results expand on the findings of Fu-
jishiro et al. (30) by demonstrating that the p38 pathway is
necessary for the insulin-stimulated downregulation of
GLUT4. Furthermore, our observation that p38 phosphor-
ylation is increased in adipocytes isolated from type 2
diabetic patients strongly supports a role for p38 to
contribute to insulin resistance and the development of
type 2 diabetes.
The 3T3-L1 adipocyte model used in this experiment
(chronic exposure to high insulin and glucose concen-
trations) mimics very closely the conditions found in
insulin-resistant human adipocytes. For example, chronic
exposure of 3T3-L1 adipocytes to insulin and glucose
results in 1) diminished insulin-stimulated glucose uptake,
2) failure of insulin to inhibit lipolysis, and 3) the loss of
IRS-1 and GLUT4 (present data; 11,31). These similarities
support the use of the 3T3-L1 adipocyte to test the role of
p38 in the regulation of IRS-1 and GLUT4. Thus we can
infer that activation of p38 observed in type 2 diabetic
adipocytes may contribute to the low levels of GLUT4
observed in those cells.
The mechanisms by which p38 may regulate GLUT4 are
currently unknown. In adipocytes from type 2 diabetic
patients, p38 phosphorylation was increased, in associa-
tion with reduced GLUT4 and IRS-1. However, unlike the
human type 2 diabetic adipocytes, insulin stimulation of
3T3-L1 adipocytes resulted in a transient increase in p38
FIG. 5. p38 phosphorylation in response to insulin in 3T3-L1 adipo-
cytes. 3T3-L1 adipocytes were pretreated without or with A-304000
before stimulation with 1 mol/l insulin for the duration indicated.
Cells were lysed, and total protein was subjected to 7.5% SDS-PAGE
and immunoblotted with the indicated antibodies. Immunoblots were
quantified using laser densitometry and are reported as integrated
optical densities in arbitrary units. The data were normalized to
unstimulated cells and are presented as means and SDs from at least
three independent experiments. *Significant difference from the basal
value (P<0.05); ‡significant difference from corresponding vehicle-
treated value (P<0.05).
C.J. CARLSON AND ASSOCIATES
DIABETES, VOL. 52, MARCH 2003 639
phosphorylation. Inhibition of p38 was able to prevent the
loss of GLUT4; thus only a transient activation of p38 may
be necessary for downregulation of GLUT4. Downregula-
tion of GLUT4 mRNA transcription in response to insulin
occurs within 2 h (25); thus it is possible that early changes
in p38 activity may be capable of modulating GLUT4
expression. GLUT4 mRNA and protein are decreased in
adipose tissue from type 2 diabetic and animal models of
insulin resistance (9,32,33) and are also diminished in 3T3-
L1 adipocytes treated with insulin (25–27), tumor necrosis
factor (TNF)-␣(34), and other factors (35,36). It has not
yet been determined if p38 regulates GLUT4 expression
through modifications of mRNA stability or mRNA tran-
scription; however, many transcription factors are known
to be downstream of the p38 pathway (13), consistent with
gene transcription as a potential mechanism.
Many regions within the GLUT4 promoter have been
identified as contributing to the downregulation of GLUT4
expression in response to insulin (37), TNF-␣(38), and
other factors (36,39). Nuclear factor 1 (NF-1) and Olf-1/
early B-cell factor (O/E) are capable of binding to an
element within the GLUT4 promoter that mediates the
insulin and cAMP-induced downregulation of GLUT4 tran-
scription (40,41). Alternatively, CCAAT-enhancer binding
proteins (C/EBP) have been shown to play important roles
in regulating GLUT4 expression during adipogenesis (42).
NF-1 becomes phosphorylated in response to insulin (43);
however, there are no data regarding the ability of p38 to
phosphorylate NF-1. p38 has been shown to be capable of
phosphorylating C/EBP (44). Thus the potential exists
for the p38 pathway to mediate repression of GLUT4
expression at the transcriptional level through directly
modifying factors such as NF-1 and C/EBP .Weare
currently pursuing further experimentation to test these
possibilities.
The current report has not addressed the nature of the
stimulus that may be responsible for the basal activation
of p38 in human adipocytes. Because the human adipo-
cytes used in the current experiment were isolated from
abdominal subcutaneous biopsies, and thus underwent a
substantial isolation process before insulin stimulation
and examination, it is likely that any systemic factors that
may have contributed to the elevated p38 phosphorylation
were removed before analysis. Thus the elevated phos-
phorylation of p38 is likely due to biochemical character-
istics intrinsic to type 2 diabetes adipocytes. Factors such
as cAMP (35), oxidative stress (36), and numerous cyto-
kines and interleukins (34) have been shown to decrease
GLUT4 protein in 3T3-L1 adipocytes and activate p38 in
other cell systems (36 and references therein). Future
experiments will be necessary to further define the cause
of p38 activation in type 2 diabetic adipocytes.
In conclusion, the current report has identified p38 as a
potential candidate for mediating the loss of GLUT4 ex-
pression observed in adipocytes from type 2 diabetic
subjects. This is based on an elevated basal phosphoryla-
tion status of p38 MAPK in human adipocytes from type 2
diabetic patients compared to healthy control subjects.
Furthermore, we have demonstrated that p38 is necessary
for the insulin-induced loss of GLUT4 expression in 3T3-L1
adipocytes. This may be of great importance because
diminished GLUT4 expression results in the development
of insulin resistance, diabetes, and many related complica-
tions (4,5). In addition, the current experiment has ruled
out the MAP kinases as contributing to the insulin-induced
loss of IRS-1 protein abundance in 3T3-L1 adipocytes.
Future work is necessary to define the mechanisms in-
volved with the activation of p38 in insulin-resistant and
diabetic adipocytes, as well as the connection between
p38 activation and regulation of GLUT4 protein abun-
dance.
ACKNOWLEDGMENTS
C.J.C. was supported by an Abbott Laboratories/North-
western University Post-Doctoral Fellowship in Diabetes.
The authors thank Dr. A. Montalvo of Northwestern
University Medical School and Dr. Silvana Gross and
members of the Department of Biochemistry, School of
Medicine, National University of Northeast (Corrientes,
Argentina) for their contributions to the project. Addition-
ally, the authors thank Dr. E. Morphew and Dr. J. Trevil-
lyan for constructive comments during preparation of the
manuscript.
REFERENCES
1. Zimmet P, Alberti KGMM, Shaw J: Global and societal implications of the
diabetes epidemic. Nature 414:782–787, 2001
2. Tamemoto H, Kadowaki T, Tobe K, Yagi T, Sakura H, Hayakawa T,
Terauchi Y, Ueki K, Kaburagi Y, Satoh S, Sekihara H, Yoshioka S, Horikoshi
H, Furuta Y, Ikawa Y, Kasuga M, Yazaki Y, Aizawa S: Insulin resistance and
growth retardation in mice lacking insulin receptor substrate-1. Nature
372:182–186, 1994
3. Araki E, Lipes MA, Patti ME, Bruning JC, Haag B 3rd, Johnson RS, Kahn
CR: Alternative pathway of insulin signalling in mice with targeted
disruption of the IRS-1 gene. Nature 372:186 –190, 1994
4. Stenbit AE, Tsao T-S, Li J, Burcelin R, Geenen DL, Factor SM, Houseknecht
K, Katz EB, Charron MJ: GLUT4 heterozygous knockout mice develop
muscle insulin resistance and diabetes. Nat Med 3:1096 –1101, 1997
5. Abel ED, Peroni O, Kim JK, Kim Y-B, Boss O, Hadro E, Minneman T,
Shulman GI, Kahn BB: Adipose-selective targeting of the GLUT4 gene
impairs insulin action in muscle and liver. Nature 409:729 –733, 2001
6. Garvey TW, Maianu L, Huecksteadt TP, Birnbaum MJ, Molina JM, and
Ciaraldi TP: Pretranslational suppression of a glucose transporter protein
causes insulin resistance in adipocytes from patients with non-insulin
dependent diabetes mellitus and obesity. J Clin Invest 87:1072–1081, 1991
7. Rondinone CM, Wang L-M, Lonnroth P, Wesslau C, Pierce JH, Smith U:
Insulin receptor substrate (IRS) 1 is reduced and IRS-2 is the main docking
protein for phosphatidylinositol 3-kinase in adipocytes from subjects with
non-insulin-dependent diabetes mellitus. Proc Natl Acad SciUSA94:
4171–4175, 1997
8. Carvalho E, Jansson P-A, Axelsen M, Eriksson JW, Huang X, Groop L,
Rondinone C, Sjostrom L, Smith U: Low cellular IRS-1 gene and protein
expression predict insulin resistance and NIDDM. FASEB J 13:2173–2178,
1999
9. Carvalho E, Jansson P-A, Nagaev I, Wenthzel A-M, Smith U: Insulin
resistance with low cellular IRS-1 expression is also associated with low
GLUT4 expression and impaired insulin-stimulated glucose transport.
FASEB J 15:1101–1103, 2001
10. Sun XJ, Goldberg JL, Qiao L-Y, Mitchell JJ: Insulin-induced insulin receptor
substrate-1 degradation is mediated by the proteasome degradation path-
way. Diabetes 48:1359 –1364, 1999
11. Pederson TM, Kramer DL, Rondinone CM: Serine/threonine phosphory-
lation of IRS-1 triggers its degradation: possible regulation by tyrosine
phosphorylation. Diabetes 50:24 –31, 2001
12. Qiao LY, Goldberg JL, Russell JC, Sun XJ: Identification of enhanced serine
kinase activity in insulin resistance. J Biol Chem 274:10625–10632, 1999
13. Widmann C, Gibson S, Jarpe MB, Johnson GL: Mitogen-activated protein
kinase: conservation of a three-kinase module from yeast to human.
Physiol Rev 79:143–180, 1999
14. Sun XJ, Rothenberg P, Kahn CR, Backer JM, Araki E, Wilden PA, Cahill DA,
Goldstein BJ, White MF: Structure of the insulin receptor substrate IRS-1
defines a unique signal transduction protein. Nature 352:73–77, 1991
15. DeFea K, Roth RA: Modulation of insulin receptor substrate-1 tyrosine
MAP KINASES AND TYPE 2 DIABETES
640 DIABETES, VOL. 52, MARCH 2003
phosphorylation and function by mitogen-activated protein kinase. J Biol
Chem 272:31400 –31406, 1997
16. Agguire V, Uchida T, Yenush L, Davis R, White MF: The c-jun NH
2
-terminal
kinase promotes insulin resistance during association with insulin recep-
tor substrate-1 and phosphorylation of ser307. J Biol Chem 275:9047–9054,
2000
17. Moxham CM, Tabrizchi A, Davis RJ, Malbon CC: jun N-terminal kinase
mediates activation of skeletal muscle glycogen synthase by insulin in
vivo. J Biol Chem 271:30765–30773, 1996
18. Miller BS, Shankavaram UT, Horney MJ, Gore ACS, Kurtz DT, Rosenzweig
SA: Activation of cjun NH
2
-terminal kinase/stress-activated protein kinase
by insulin. Biochem 35:8769 –8775, 1996
19. Sweeney G, Somwar R, Ramlal T, Volchuk A, Ueyama A, Klip A: An
inhibitor of p38 mitogen-acitvated protein kinase prevents insulin-stimu-
lated glucose transport but not glucose transporter translocation in 3T3–L1
adipocytes and L6 myotubes. J Biol Chem 274:10071–10078, 1999
20. Desbois-Mouthon C, Blivet-Van Eggelpoel M-J, Auclair M, Cherqui G,
Capeau J, Caron M: Insulin differentially regulates SAPKs/JNKs and ERKs
in CHO cells overexpressing human insulin receptors. Biochem Biophys
Res Comm 243:765–770, 1998
21. Kayali AG, Austin DA, Webster NJG: Stimulation of MAPK cascades by
insulin and osmotic shock: lack of an involvement of p38 mitogen-
activated protein kinase in glucose transport in 3T3–L1 adipocytes. Dia-
betes 49:1783–1793, 2000
22. Cusi K, Maezono K, Osman A, Pendergrass M, Patti ME, Pratipanawatr T,
Defronzo RA, Kahn CR, Mandarino LJ: Insulin-resistance differentially
affects the PI 3-kinase- and MAP kinase-mediated signaling in human
muscle. J Clin Invest 105:311–320, 2000
23. Krook A, Bjornholm M, Galuska D, Jiang XJ, Fahlman R, Myers MG Jr,
Wallberg-Henriksson H, Zierath JR: Characterization of signal transduction
and glucose transport in skeletal muscle from type 2 diabetic patients.
Diabetes 49:284 –292, 2000
24. Smith U, Sjostrom L, Bjornstorp P: Comparison of two methods for
determining human adipose cell size. J Lipid Res 13:822–824, 1972
25. Flores-Riveros JR, McLenithan JC, Ezaki O, Lane MD: Insulin down-
regulates expression of the insulin-responsive glucose transporter
(GLUT4) gene: effects on transcription and mRNA turnover. Proc Natl
Acad SciUSA90:512–516, 1993
26. Thomson MJ, Williams MG, Frost SC: Development of insulin resistance in
3T3–L1 adipocytes. J Biol Chem 272:7759 –7764, 1997
27. Yu Z-W, Buren J, Enerback S, Nilsson E, Samuelson L, Eriksson JW: Insulin
can enhance GLUT4 gene expression in 3T3–F442A cells and this effect is
mimicked by vanadate but counteracted by cAMP and high glucose:
potential implications for insulin resistance. Biochim Biophys Acta 1535:
174 –185, 2001
28. Cuenda A, Rouse J, Doza YN, Meier R, Cohen P, Gallagher TF, Young PR,
Lee JC: SB203580 is a specific inhibitor of a MAP kinase homologue which
is stimulated by cellular stresses and interleukin-1. FEBS Lett 364:229 –233,
1995
29. Somwar R, Koterski S, Sweeney G, Sciotti R, Djuric S, Berg C, Trevillyan
J, Scherer PE, Rondinone CR, Klip A: A dominant-negative p38 MAPK
mutant and novel selective inhibitors of p38 MAPK reduce insulin-
stimulated glucose uptake in 3T3–L1 adipocytes without affecting GLUT4
translocation. J Biol Chem 277:50386 –50395, 2002
30. Fujishiro M, Gotoh Y, Katagiri H, Sakoda H, Ogihara T, Anai M, Onishi Y,
Ono H, Funaki M, Inukai K, Fukushima Y, Kikuchi M, Oka Y, Asano T:
MKK6/3 and p38 MAPK pathway activation is not necessary for insulin-
induced glucose uptake, but regulates glucose transporter expression.
J Biol Chem 276:19800 –19806, 2001
31. Berg CE, Lavan BE, Rondinone CM: Rapamycin partially prevents insulin
resistance induced by chronic insulin treatment. Biochem Biophys Res
Commun 293:1021–1027, 2002
32. Kramer D, Shapiro R, Adler A, Bush E, Rondinone CM: Insulin-sensitizing
effect of rosiglitazone (BRL-49653) by regulation of glucose transporters in
muscle and fat of zucker rats. Metabolism 50:1294 –1300, 2001
33. Charron MJ, Katz EB, Olson AL: GLUT4 gene regulation and manipulation.
J Biol Chem 274:3253–3256, 1999
34. Stephens JM, Pekala PH: Transcriptional repression of the GLUT4 and
C/EBP genes in 3T3–L1 adipocytes by tumor necrosis factor-alpha. J Biol
Chem 266:21839 –21845, 1991
35. Kaestner KH, Flores-Riveros JR, McLenithan JC, Janicott M, Lane MD:
Transcriptional repression of the mouse insulin-responsive glucose trans-
porter (GLUT4) gene by cAMP. Proc Natl Acad SciUSA88:1933–1937,
1991
36. Pessler D, Rudich A, Bashan N: Oxidative stress impairs nuclear proteins
binding to the insulin responsive element in the GLUT4 promoter. Diabe-
tologia 44:2156 –2164, 2001
37. Cooke DW, Lane MD: A sequence element in the GLUT4 gene that
mediates repression by insulin. J Biol Chem 273:6210 –6217, 1998
38. Jain R, Police S, Phelps K, Pekala PH: Tumour necrosis factor-alpha
regulates expression of the CCAAT-enhancer-binding proteins (C/EBPs)
alpha and beta and determines the occupation of the C/EBP site in the
promoter of the insulin-responsive glucose-transporter gene in 3T3–L1
adipocytes. Biochem J 338:737–743, 1999
39. Flores-Riveros JR, Kaestner KH, Thompson KS, Lane MD: Cyclic AMP-
induced transcriptional repression of the insulin-responsive glucose trans-
porter (GLUT4) gene: identification of a promoter region required for
down-regulation of transcription. Biochem Biophys Res Comm 194:1148 –
1154, 1993
40. Dowell P, Cooke DW: Olf-1/Early B-cell factor (O/E) is a regulator of
GLUT4 gene expression in 3T3 L1 adipoctytes. J Biol Chem 277:1712–1718,
2002
41. Cooke DW, Lane MD: Transcription factor NF1 mediates repression of the
GLUT4 promoter by cyclic-AMP. Biochem Biophys Res Comm 260:600 –
604, 1999
42. Wu Z, Rosen ED, Brun R, Hauser S, Adelmant G, Troy AE, McKeon C,
Darlington GJ, Spiegelman BM: Cross-regulation of C/EBPalpha and
PPARgamma controls the transcriptional pathway of adipogenesis and
insulin sensitivity. Mol Cell 3:151–158, 1999
43. Cooke DW, Lane MD: The transcription factor nuclear factor 1 mediates
repression of the GLUT4 promoter by insulin. J Biol Chem 274:12917–
12924, 1999
44. Engelman JA, Lianti MP, Schere PE: Specific inhibitors of p38 mitogen-
activated protein kinase block 3T3–L1 adipogenesis. J Biol Chem 273:
32111–32120, 1998
C.J. CARLSON AND ASSOCIATES
DIABETES, VOL. 52, MARCH 2003 641
