Selective Serotonin Reuptake Inhibitors (SSRIs) Inhibit Insulin Secretion and Action in Pancreatic β Cells IMI CONFIDENTIAL J. Biol. Chem. 2013, 288:5682-5693,

Selective Serotonin Reuptake Inhibitors (SSRIs) Inhibit Insulin Secretion and

Action in Pancreatic β Cells

IMI CONFIDENTIAL

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。


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。


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 .


Materials and Methods

Mice

Male C57BL/6 mice.

Cells

Mouse insulinoma (Min6) cells

Primary mice/human islet cells


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


1. Sertraline Inhibits Insulin-induced Tyr Phosphorylation of IRS-2 and Its Coupling with Downstream Effectors

Results

Fig. 1


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


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.


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.


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.


Results

Fig. 5

Results of IP


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.


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.


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.


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, …


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.


Results


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


Results

sertraline iNOS ER stress/UPR apoptosis


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Selective Serotonin Reuptake Inhibitors (SSRIs) Inhibit Insulin Secretion and Action in Pancreatic β Cells IMI CONFIDENTIAL J. Biol. Chem. 2013, 288:5682-5693,