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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 14, 2000 as doi:10.1096/fj.00-0414fje. |
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* Laboratory of Molecular Medicine, Department of Internal Medicine, University of Rome-‘Tor Vergata’, 00133 Rome, Italy;
IDI-IRCCS, Biochemistry Laboratory, Department of Experimental Medicine, University of Rome-‘Tor Vergata’, Rome, Italy; and
Department of Endocrinology and Metabolism, University of Pisa, 56100 Pisa, Italy
2Correspondence: Dipartimento di Medicina Interna, Università di Roma-‘Tor Vergata’, Via di Tor Vergata, 135, 00133 Roma, Italy. E-mail: sesti{at}uniroma2.it
SPECIFIC AIMS
Molecular scanning of the human IRS-1 gene revealed that diabetic and prediabetic carriers of a common polymorphism causing a Gly to Arg change at codon 972 (Arg972 IRS-1) are characterized by a low fasting plasma concentration of insulin and C peptide. We have directly addressed the effect of Arg972 IRS-1 variant in human pancreatic islet survival and characterized a downstream signaling pathway involved in anti-apoptotic effects of insulin in pancreatic ß cells.
PRINCIPAL FINDINGS
1. Survival of human pancreatic islets and RIN ß cells
expressing Arg972 IRS-1
To investigate directly whether the
Arg972 IRS-1 variant affects islet cells
survival, we analyzed pancreatic islets isolated from three donors
heterozygous for the Arg972 and six donors
carrying wild-type IRS-1. Immunofluorescence analysis revealed
increased apoptosis induced by serum deprivation of insulin-positive
ß cells in islets from the carriers of Arg972
IRS-1 (Fig. 1A
, lower panel) as compared with wild-type (Fig. 1A
, upper panel). To confirm this finding quantitatively, we
used flow cytometry to determine the number of hypodiploid events after
propidium iodide staining of fixed cells. Under basal conditions,
islets from carriers of Arg972 IRS-1 variant
showed a twofold increase in the number of apoptotic cells as compared
with wild-type (45±8% vs. 16±4%, respectively; P<0.009)
(Fig. 1B
). Apoptosis induced by serum deprivation was also
increased in islets from the carriers of Arg972
IRS-1 as compared with wild-type (65±12% vs. 21±5%, respectively;
P<0.009) (Fig. 1B
). To determine whether insulin
protects islets from apoptosis, we examined the effect of insulin on
the survival of human islets subjected to serum deprivation. Addition
of insulin at the time of serum deprivation was less effective at
inhibiting apoptosis in islet cells from Arg972
IRS-1 carriers as compared with wild-type (57±15 vs. 13±2%,
respectively; P
0.006) (Fig. 1B
). We next
compared IRS-1-associated PI 3-kinase activity in islet cells from
carriers of Arg972 IRS-1 variant and wild-type
carriers. Compared with wild-type carriers, PI 3-kinase activity
associated with IRS-1 in islet cells from Arg972
IRS-1 carriers was decreased by 17% (P<0.05) in the basal
state and by 36% upon stimulation with insulin (P<0.04)
(data not shown). To further define the molecular mechanisms of the
increased susceptibility to apoptosis caused by
Arg972 IRS-1 variant, we used RIN ß cell lines
stably expressing equal amounts of either wild-type IRS-1 (RIN-WT) or
Arg972 IRS-1 (RIN-Arg972).
We performed flow cytometry using annexin V/PI double staining with
fresh unfixed cells. The dot plot from RIN-WT cells did not show
relevant positiveness to annexin V, whereas
RIN-Arg972 cells showed marked positiveness to
annexin V, which revealed higher apoptosis than RIN-WT cells (Fig. 1C
).
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2. PI-3-kinase activity and Akt phosphorylation, but not MAPK
activation, are reduced in RIN ß cells expressing wild-type IRS-1 or
Arg972 IRS-1
The extent of basal and insulin-stimulated tyrosine
phosphorylation of IRS-1 was similar in both RIN-WT and
RIN-Arg972 cells (Fig. 2A
). As compared with RIN-WT cells, IRS-1-associated PI-3
kinase activity in RIN-Arg972 cells was reduced
by 20% (P < 0.05) in the basal state and by 50% upon
stimulation with insulin for 2 min, 10 min, or 20 min (P <
0.04) (Fig. 2A
). Basal Ser473-Akt
phosphorylation was decreased by 20% in
RIN-Arg972 compared with RIN-WT cells
(P < 0.03) (Fig. 2A
). Insulin-induced
Ser473-Akt phosphorylation was reduced in
RIN-Arg972 by 50% (P < 0.001) (Fig. 2A
). Basal and insulin-stimulated MAPK phosphorylation did
not differ between RIN-WT and RIN-Arg972 cells
(data not shown).
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3. BAD phosphorylation and association with Bcl-XL or
14–3-3 protein in RIN ß cells expressing wild-type IRS-1 or
Arg972 IRS-1
Compared with RIN-WT cells, BAD phosphorylation at
Ser136 in RIN-Arg972 was
decreased by 30% (P < 0.04) in the basal state, and by
50% upon stimulation with insulin (P < 0.004) (Fig. 2A
). By contrast, BAD phosphorylation at
Ser112 did not differ between RIN-WT and
RIN-Arg972 cells (Fig. 2A
).
Immunoblotting analysis with anti-BAD antibody of cells lysates
immunoprecipitated with Bcl-XL revealed that
association of BAD with Bcl-XL was increased in
RIN-Arg972 cells as compared with RIN-WT cells
under both basal condition and in response to insulin (Fig. 2B
). Consistent with these results, immunoblotting analysis
with anti-BAD antibody of cells lysates immunoprecipitated with
anti-14-3-3 antibody revealed that association of BAD with 14-3-3 was
reduced in RIN-Arg972 cells in the basal state
and upon insulin stimulation (Fig. 2B
).
4. Caspase-9 and caspase-3 activity in RIN ß cells
expressing wild-type IRS-1 or Arg972 IRS-1
We investigated whether the increased association of BAD
with Bcl-XL observed in RIN ß cells that
express Arg972 IRS-1 variant resulted in enhanced
proteolytic activity of both caspase-9 and caspase-3. We observed that
caspase-9 and caspase-3 activity was markedly increased in
RIN-Arg972 cells as compared with RIN-WT cells
either under basal conditions or after serum deprivation (Fig. 2C
).
CONCLUSIONS AND SIGNIFICANCE
In the present study, we provide evidence that the
Arg972 IRS-1 variant, which is more common among
type 2 diabetic patients, impairs pancreatic ß cell survival, which
may contribute to the relative insulin deficiency observed in carriers
of this variant. We document for the first time in human tissues that
pancreatic islets isolated from carriers of
Arg972 IRS-1 exhibit impaired IRS-1-associated PI
3-kinase activity and increased apoptosis and that they appear
resistant to the anti-apoptotic effect of insulin as compared with
wild-type controls. The same results were reproduced in RIN rat ß
cell lines stably expressing either wild-type IRS-1 or
Arg972 IRS-1 variant. Using these cell lines, we
have characterized the downstream signaling pathway by which
Arg972 IRS-1 variant impairs pancreatic ß cell
survival. RIN cells expressing Arg972 IRS-1
exhibited a marked impairment in the sequential activation of PI
3-kinase and Akt as compared with RIN cells expressing wild-type IRS-1.
The decrease in Arg972 IRS-1-associated PI
3-kinase activity was not caused by defects in IRS-1 tyrosine
phosphorylation, but by a defective activation of PI 3-kinase by IRS-1.
Recent studies have demonstrated that Akt promotes survival by acting
at multiple sites. These actions include phosphorylation and the
subsequent inactivation of components of the cell death machinery
possessing the RXRXXS/T consensus sequence. Among these is BAD, a
member of the BCL-2 family of proteins that are critical regulators of
apoptosis. In mammalian cells, BAD exerts its proapoptotic effect in
part by sequestering Bcl-XL in nonfunctional
heterodimers, whereas upon phosphorylation BAD dissociates from
Bcl-XL and interacts with cytoplasmic 14-3-3
phosphoserine binding protein. The released
Bcl-XL then promotes cell survival by blocking
the caspase protease cascade. Consistent with this model are our
findings that, in RIN cells expressing Arg972
IRS-1, both basal and insulin-stimulated BAD phosphorylation on serine
136 was reduced as compared with RIN cells expressing wild-type IRS-1,
which results in an increased binding to Bcl-XL
instead of to 14-3-3 protein. These data suggest that impaired
activation of the insulin signaling pathway involving sequential
induction of IRS-1-associated PI 3-kinase activity, Akt activity, and
BAD phosphorylation is responsible for the increased apoptosis observed
in pancreatic islets isolated from carriers of
Arg972 IRS-1 (Fig. 3
). Thus, a selective resistance of pancreatic ß cells to insulin may
lead to impairment in the control of the programmed cell death
machinery when apoptotic stimuli is exerted. The impaired cell survival
observed in human islets expressing Arg972 IRS-1
variant contrasts with the ß cell hyperplasia observed in mice with
disruption of the IRS-1 gene. This disparity may be due to
several factors. First, compensatory overexpression of IRS-2 in mice
lacking IRS-1 may mask the abnormal function of the ß cells. Second,
a complex interplay exists between IRS-1 and IRS-2 in the regulation of
downstream signaling, including the PI 3-kinase pathway. For example,
it has been reported that IRS-1-associated PI 3-kinase activity is
decreased markedly in skeletal muscle of mice lacking IRS-2, whereas
IRS-2-associated PI 3-kinase activity is enhanced markedly in mice
lacking IRS-1. This finding raises the possibility that alterations in
the balance between these signaling molecules may contribute to ß
cell dysfunction. Moreover, we have observed that the activity of the
IRS-2/PI 3-kinase pathway is not modified in the presence of the
Arg972 IRS-1 variant. Finally, of course, we
cannot exclude differences between species in terms of the relative
role of IRS-1 and IRS-2 in ß cell function. The data in the present
study provide evidence of a functional role for insulin in human islet
survival and suggest that polymorphism in insulin signaling proteins,
such as the naturally occurring Arg972 IRS-1
variant, may confer susceptibility to apoptosis of pancreatic ß
cells.
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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.00-0414fje To cite this article, use (November 14, 2000) FASEB J. 10.1096/fj.00-0414fje 
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