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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 5, 2001 as doi:10.1096/fj.00-0435fje. |
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The Lundberg Laboratory for Diabetes Research, Department of Internal Medicine, Sahlgrenska University Hospital, Göteborg, Sweden
2Correspondence: The Lundberg Laboratory for Diabetes Research, Department of Internal Medicine, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden. E-mail: ulf.smith{at}medic.gu.se
SPECIFIC AIMS
Lowexpression of the key docking protein insulin receptor substrate-1
(IRS-1) has been found in fat cells from type 2 diabetic subjects as
well as a cohort of non-diabetic but insulin-resistant individuals. In
this study, we examined the in vivo phenotypes as well as
the insulin signaling and action in cells from healthy subjects
characterized as having normal or low (
50% of normal) IRS-1
expression.
PRINCIPAL FINDINGS
1. Phenotypic characterizations
Subjects with low IRS-1 were slightly older than the group with
normal IRS-1 expression. They had significantly larger fat cells and
waist/hip circumference ratio (WHR), which suggests an abdominal fat
distribution. Because of a predominance of males in the low IRS-1 group
(18/20), these individuals were also compared with the male controls
only. However, the differences in WHR and fat cell size still differed
significantly. A clear overrepresentation of individuals with a known
genetic predisposition for diabetes in the low IRS-1 group (14/20;
70%) was apparent, whereas 6/20 (30%) had no known genetic
predisposition.
2. Insulin signaling and protein expression
As shown in
Fig. 1
, the low IRS-1 group had 
65% lower IRS-1
protein expression than the control group (p<0.001).
However, the expression of other examined intracellular proteins, such
as p85, IRS-2, PKB/Akt, and MAP kinase, did not differ between the
groups.
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The reduced IRS-1 expression was also associated with a marked
impairment in insulin-stimulated PI3-kinase activity in total
antiphosphotyrosine immunoprecipitates (Fig. 1)
. The average reduction
in PI3-kinase activity in four deficient versus nondeficient cells was
70%. Thus, low IRS-1 expression is also associated with low
cellular insulin-stimulated PI3-kinase activity. We also examined the
consequences of the low PI3-kinase activity on the downstream signaling
in response to insulin. PKB/Akt serine phosphorylation was reduced
60%–70% (Fig. 1)
.
3. Glucose transport and GLUT4 expression
Maximally insulin-stimulated glucose transport was significantly
lower (60% reduction) in IRS-1-deficient cells
(Fig. 2
), whereas basal (nonstimulated) glucose uptake was
similar. This impairment in activation of glucose transport by insulin
could be due to the impairment in insulin signaling (Fig. 1)
or to a
reduced GLUT4 protein expression. This condition was examined by adding
1 µM okadaic acid, a PP1 and 2A threonine/serine phosphatase
inhibitor that exerts a full insulin-like effect which is PI3-kinase
independent, on glucose transport in human fat cells. Okadaic acid
improved but did not restore the insulin-stimulated glucose transport.
This finding suggests that GLUT4 protein expression may be reduced, and
Fig. 2
shows that low cellular IRS-1 expression is associated with
60% reduction in GLUT4 expression.
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4. Gene expression of IRS-1 and GLUT4
Real-time RT-PCR was used to assay mRNA levels for IRS-1 and GLUT4
because large tissue samples were generally not obtained in these
nonobese individuals. For comparison, we also assayed the gene
expression in fat cells from five type 2 diabetic subjects.
Both IRS-1 and GLUT4 gene expression levels were significantly lower in
diabetic cells when compared with the control group (50% and
60% reduction, respectively; P<0.02 and
P<0.005). The mRNA levels were reduced 40% for GLUT4
(P<0.05) in the IRS-1 deficient cells when compared with
nondeficient cells. IRS-1 mRNA was reduced 20% in the deficient
cells but this finding was not statistically significant because of
large interindividual variations in both groups.
CONCLUSIONS
The present study is an extension of our previous investigation,
in which we found that low cellular IRS-1 expression, defined as 50%
of normal, identifies individuals with insulin resistance and various
markers of the insulin resistance syndrome in healthy, nondiabetic
subjects. Enlarging the study to include about twice the number of
nonobese subjects confirmed our previous result that a low cellular
IRS-1 expression occurs approximately two- to threefold more frequently
in individuals with a genetic predisposition for type 2 diabetes.
Furthermore, this finding is associated with a larger waist/hip
circumference ratio, which is a well-established marker of insulin
resistance and propensity for type 2 diabetes, also in this nonobese
group.
The novel salient findings of the present study are that low IRS-1 expression is associated with a marked impairment in downstream insulin signaling, including PI3-kinase activation; serine phosphorylation of PKB/Akt and maximally insulin-stimulated glucose transport is impaired; and GLUT4 protein expression is also markedly reduced. In fact, all these perturbations are similar, both in terms of extent and proteins involved, to what is seen in fat cells from subjects with manifest type 2 diabetes. From this point of view, it appears that healthy individuals with a low IRS-1 expression also exhibit an adipose tissue with diabetic traits in terms of insulin signaling and response. This concept needs to be extended by characterizing other aspects of adipose tissue metabolism and function such as the lipolytic response and the antilipolytic effect of insulin.
The concept that the adipose tissue already exhibits several diabetic
traits in these healthy subjects is indeed astonishing. Similar to
diabetic cells, PKB/Akt serine phosphorylation was impaired even in the
presence of a supramaximal insulin concentration. Whether this finding
is due to the expression of a truncated PKB/Akt lacking the serine
phosphorylation site and/or to the 70% reduction in
insulin-stimulated PI3-kinase activity remains to be established.
However, this trait appears to be a consistent finding with fat cells
from different insulin-resistant states.
A low IRS-1 expression was also associated with a low GLUT4 expression
and a reduced insulin-stimulated glucose transport. Okadaic acid, which
activates glucose transport and PKB/Akt in human fat cells to a similar
extent as insulin but in a PI3-kinase independent manner, did not
restore the glucose transport to normal. These data suggest that low
GLUT 4 expression was the major reason for the impaired
insulin-stimulated glucose transport in these cells. Furthermore, the
data suggest the possibility of a coordinate regulation of IRS-1 and
GLUT4. The low mRNA expression for both IRS-1 and GLUT4 in the diabetic
cells when compared with the control group suggests that this
expression occurs at the level of gene transcription. Similarly, mRNA
for GLUT4 was reduced significantly in IRS-1-deficient cells, whereas
the difference in IRS-1 expression was not significant due to large
interindividual variations in both groups. One possibility is that
deficient cells have an elevated production of TNF-
, which has been
reported to lower both IRS-1 and GLUT4 expression. This and other
possibilities are the focus of ongoing studies in our laboratory.
A key question raised by the present data is whether the reduced IRS-1 and GLUT4 expression in the adipose tissue could be causally related to the insulin resistance and type 2 diabetes or whether it is a marker of a common pathogenetic mechanism(s). Support for the first possibility is the recent observation that adipose-specific GLUT4 gene disruption in mice is associated with a marked insulin resistance. Furthermore, total IRS-1 gene disruptionin mice leads to a marked insulin resistance. No current animal model shows tissue-specific IRS-1 gene knockout to indicate the relative role of the adipose tissue versus muscle and liver for the insulin resistance.
The present study has shown that a low IRS-1 expression is associated with a marked impairment in insulin-stimulated PI3-kinase activity and downstream insulin signaling. Furthermore, GLUT4 protein expression and insulin-stimulated glucose transport are also reduced, which suggests the possibility of a coordinate regulation of IRS-1 and GLUT4, probably at the level of gene transcription. Whether these perturbations are causally related to insulin resistance or whether they reflect a common pathogenetic mechanism(s) remains to be established. However, the abnormalities seen in this group of healthy subjects are very similar to those seen in cells from type 2 diabetic subjects. Thus, it appears that diabetic traits can be present in the adipose tissue long before type 2 diabetes develops.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0435 to cite
this article, use (February 6, 2001) FASEB J. 10.1096/fj.00-0435fje 
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