selective serotonin reuptake inhibitors (ssris) inhibit insulin secretion and action in pancreatic...
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Selective Serotonin Reuptake Inhibitors (SSRIs) Inhibit Insulin Secretion and
Action in Pancreatic β Cells
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J. Biol. Chem. 2013, 288:5682-5693, IF=4.6
Xu Zhenjie2014.11.13
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Introduction
Selective serotonin reuptake inhibitors (SSRIs) are antidepressants used
for the treatment of mood and anxiety disorders. SSRIs block only the
serotonin (血清素 /5-羟色胺 ) transporter.
such as paroxetine (帕罗西汀 ), fluoxetine (氟西汀 ), sertraline (舍曲林 ).
Side effect of SSRIs: Long term use of SSRIs is associated with an
increased risk of diabetes. But little is known about the pathophysiology
of SSRIs as direct inducers of diabetes.
Insulin receptor substrate ( IRSs):能够被激活的胰岛素受体酪氨酸激酶作用的底物,磷酸化的 IRSs能够结合并激活下游效应物。
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Introduction
已知有三种 IRSs。第一种是 IRS1,
是一种蛋白质 , 磷酸化后可同多种效应物结合 ,包括 :PI(3)K、 Syp、Nck、 GRB2(growth factor
receptor-bound protein 2)。第二种是 Shc, Shc的酪氨酸被磷酸化后能够同 GRB2结合 ,然后激活 Ras,
触发细胞的增殖。第三种底物是IRS2, IRS2的酪氨酸被磷酸化后能够同磷脂酰肌醇 -3-激酶( PI3K)结合,将该酶激活并影响磷脂的代谢。
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Introduction
IRS proteins play a key role in growth and survival of pancreatic β cells.
Irs2 -/- mice develop diabetes 8–10 weeks after birth due to reduced β cell
mass and impaired β cell function. Conversely, cell-specific expression of
Irs2 promotes β cell growth, survival, and insulin secretion and prevents
diabetes in Irs2 -/- mice, obese mice, and streptozotocin-treated mice .
IRS-2-mediated signaling also protects β cells from undergoing
apoptosis .
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Materials and Methods
Mice
Male C57BL/6 mice.
Cells
Mouse insulinoma (Min6) cells
Primary mice/human islet cells
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Materials and Methods
Immunoprecipitation
Western blot
siRNA transfection
Mass spectrometry
Real-time PCR
Adenoviral infection
Flow cytometry
Glucose-stimulated Insulin Secretion (GSIS)
ELISA
Cellular Reducing Power
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1. Sertraline Inhibits Insulin-induced Tyr Phosphorylation of IRS-2 and Its Coupling with Downstream Effectors
Results
Fig. 1
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Fig. 1
Results
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Fig. 2
Results
Sertraline Inhibits Insulin-induced Tyr
Phosphorylation of IRS-2 and Its Coupling with
Downstream Effectors
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2. Activation of MAPK Family Members and GSK3β by SSRIs
Results
Fig. 3
The stress-activated kinase, JNK, inhibits the activity of IRS proteins through Ser/Thr phosphorylation.
An inverse correlation existed
between Tyr phosphorylation
of IRS-2 and the activation of
JNK.
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Fig. 4
Results
To explore the effects of SSRIs on other members of the MAPK family,…
Sertraline inhibited the basal phosphorylation of GSK3 in a time- dependentmanner, thus increasing its activity.
accompanied the decreased Tyr phosphorylation of IRS-2 and the reduced activation of its downstream effector Akt.
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3. siRNAs to GSK3β, but Not to JNK, Attenuate the Inhibitory Effects of SSRIs on Insulin Signaling
Results
Fig. 5
To directly evaluate the role of JNK and GSK3 as mediators of the inhibitory effects of sertraline,..
The reduced expression of GSK3 eliminated
the inhibitory effects of sertraline on Tyr
phosphorylation of IRS-2 (and the activation of
Akt). But the siRNAs to JNK failed to do so.
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Results
Fig. 5
Results of IP
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4. Sertraline Induces Ser/Thr Phosphorylation of IRS-2
Results
Fig. 6IP, MASS
All of the above results were consistent with the hypothesis that sertraline induces Ser/Thr phosphorylation of IRS-2. To provide a direct proof to this concept,…
Sertraline selectively increased the phosphorylation of at least 15 Ser/Thr residues.
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5. Effects of SSRIs on Insulin Signaling in Isolated Islets
Results
Fig. 7
Isolated mouse islets, infected with a recombinant adenovirus harboring Myc-IRS-2WT.
Pretreatment with sertraline did not affect the cellular content of IRS and Akt proteins. However, it decreased the insulin-induced Tyr phosphorylation of IRS-2 and Akt.
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6. SSRIs Inhibit Glucose-stimulated Insulin Secretion (GSIS)
Results
Proper action of IRS proteins is essential for β cell function and insulin secretion. Therefore, we
examined whether sertraline affects glucose-stimulated insulin secretion.
Fig. 8
Min6 cells (A), mouse (B), and human (C) pancreatic islets
Cells treated with sertraline exhibited normal basal insulin secretion, however their GSIS was reduced. In contrast, glucose-independent secretion, induced by KCl and Arg remained unaffected.
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Fig. 9
Results
Sertraline at 30 μM markedly reduced the glucose-induced increase in cellular reducing power. Sertraline however had no effect on the basal cellular reducing power. Similar results were obtained with mouse islets (data not shown).
Min6 cellsShort term, 2h
LiCl is a specific inhibitor of GSK3.
The glucose-induced increase in cellular reducing power is a key step in insulin secretion. To determine whether the reduced GSIS could be attributed to a reduction in cellular reducing power, …
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7. Long Term Effects of Sertraline on Cell Viability
Fig. 10
Results
UntreatedLiClBIO-X
Non-targeting control siRNAsiRNA to JNK
Min6 cellsLong term, 16h
Although short term treatment with sertraline did not affect cell viability (Fig. 9), long term (16-h) incubation with the drug induced an apoptotic process and death of Min6 cells. Cellular reducing power also decreased in the long term presence of sertraline.GSK3β inhibitor LiCl and BIO-X eliminated the effect of Sertraline.Silencing of JNK partially eliminated the effect of Sertraline.
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Results
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8. Sertraline Induces an ER Stress and the UPR in Min6 Cells
Results
To determine whether sertraline triggers an ER stress response, the effects of sertraline on ATF4
(activating transcriptional factor 4) and CHOP (C/EBP Homology Protein, a stress-induced
transcription factor), key elements along the unfolded protein response (UPR) signaling pathway,
were analyzed.
thapsigargin, a classical inducer of ER stress
2-h treatment did not affect the mRNA
levels of ATF4 or CHOP.
16 h treatment significantly increased
mRNA levels of ATF4 and CHOP.
Fig. 11
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Results
sertraline iNOS ER stress/UPR apoptosis
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